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Archive for the ‘3D Plotting Scaffolds’ Category


Mid Atlantic LRIG 22nd Annual Technology Showcase: Agenda on 3D Bioprinting on Wednesday, May 11, 2016 at Holiday Inn, 195 Davidson Avenue, Somerset, NJ

Reporter: Stephen J. Williams, Ph.D.

 

Symposium Speakers and Topics:

Human Organoids
Hatem E. Sabaawy-Director, Production GMP Facility for Cell and Gene Therapy, RBHS-Robert Wood Johnson Medical School, Rutgers Cancer Institute of New Jersey

Intestinal Organoids for Drug Discovery
Richard Visconti-Associate Principal Scientist, Cellular Pharmacology, Merck Research Laboratories, Kenilworth,  New Jersey

3D Bioprinting
Elizabeth Wu-President, WuZenTech, Edison, New Jersey

Building  Your Brand  Through LinkedIn
Stan Robinson, Jr., LinkedIn Consultant, Helping Professionals with Social Selling, Personal Branding

Register at EventBrite here: https://www.eventbrite.com/e/mid-atlantic-22nd-annual-technology-and-exhibition-tickets-21359945171 

To sign up to be an LRIG member or update your profile, please visit us at http://lrig.org
Hoping to see you on May 11th.
Reserve your spot today!

 

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GE’s large scale 3D cookbook

Curator: Larry H. Bernstein, MD, FCAP

 

 

Major Laser: These Scientists Are Writing the 3D-Printing Cookbook for GE

Additive manufacturing engineer Brian Adkins in full gear is preparing a DMLM machine for printing. (Photo credit: GE Reports/Chris New)

It would be a stretch to say that Joe Vinciquerra is the Julia Child of GE. But Vinciquerra, the manager of the newly formed Additive Materials Lab at GE Global Research, is creating a cookbook that will likely impact manufacturing across GE the same way “Mastering the Art of French Cooking” shook up American kitchens.

Additive manufacturing, commonly known as 3D printing, is exploding right now. GE estimates that by 2025, more than 20 percent of new products will involve additive processes of some kind. But there’s no cookbook that standardizes the recipes, which have oodles of parameters that determine the properties of the final part.

“It’s like baking a cake. You need to start with the right recipe, then you need to have the right ingredients and the right oven,” Vinciquerra says. “A cup of materials science, a tablespoon of design and a whole lot of machine-control strategies must come together and yield perfection.”

Technologies like direct metal laser melting (DMLM), for example, can involve several lasers as powerful as 1 kilowatt—enough to burn a hole in a wall—fusing as many as 1,250 layers of fine superalloy powder into the desired shape. Some large builds can take days to finish.

support block with 3D printed parts inside a DMLM printed in Pittsburgh. (Photo credit: GE Reports/Chris New)

Last week, GE opened a new industrial-scale 3D-printing center in Pittsburgh, Pennsylvania. It will work closely with Vinciquerra’s team, test their findings and get GE factories quickly cooking with additive.

His team has already started testing and tabling the powdered materials used in additive manufacturing and their properties. “We want to know how they come together, how they affect each other and what machines and processes are best suited for them,” Vinciquerra says. “It’s just like a gourmet recipe. We need to know how our ingredients are going to react in a mixer or an oven. And what changes can we make to those ingredients, the mixer or the oven to produce a more palatable dish?”

The team is pulling in expertise from other labs on the GE Global Research campus in Niskayuna, New York, including scientists focusing on nanomaterials, microstructures and machine design. The company calls the cross-pollination of know-how the GE Store.

inciquerra (right) and Andy Deal, a metallurgist in the Additive Materials Lab are loading sets of sample 3D printed metal parts in a vacuum oven for post-processing at GE Global Research. (Photo credit: GE Global Research.)      http://www.pharmpro.com/sites/pharmpro.com/files/styles/content_body_image/public/embedded_image/2016/04/Major%20Laser_GE%20Reports_3.jpg?itok=GSQMNM4L

 

GE materials scientists are no strangers to new materials. They spent two decades developing light- and heat-resistant materials called ceramic matrix composites that outperform even the most advanced superalloys and make jet engines and gas turbines lighter and more efficient. But additive materials live in a different universe. “With additive, you can design as you go and create architectures that cannot be manufactured by any other means,” Vinciquerra says.

He says that GE engineers can already design components with sophisticated, performance-enhancing features previously unattainable by any other means of manufacturing. The next-generation LEAP jet engine—developed by CFM International, a joint venture between GE Aviation and France’s Snecma (Safran)—uses 3D-printed fuel nozzles, which are 25 percent lighter and five times more durable. They used to be made from 18 separate parts and now they come in one piece. A year ago, the Federal Aviation Administration (FAA) approved a fist-sized housing for a sensor as the first 3D-printed part to fly inside GE commercial jet engines.

“This is just the beginning,” Vinciquerra says. “Someday, we may even be able to combine materials together in ways previously not possible to unlock new capabilities that never existed. Can I create a new class of materials that open the design envelope and push the limits of durability and heat resistance beyond what we thought was even possible? We’re going to find out.”

To read the original story, published on GE Reports, click here

 

 

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3D revolution and tissue repair

Curator: Larry H. Bernstein, MD, FCAP

 

 

Berkeley Lab captures first high-res 3D images of DNA segments

DNA segments are targeted to be building blocks for molecular computer memory and electronic devices, nanoscale drug-delivery systems, and as markers for biological research and imaging disease-relevant proteins

In a Berkeley Lab-led study, flexible double-helix DNA segments (purple, with green DNA models) connected to gold nanoparticles (yellow) are revealed from the 3D density maps reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography (IPET). Projections of the structures are shown in the green background grid. (credit: Berkeley Lab)

An international research team working at the Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images of double-helix DNA segments attached at either end to gold nanoparticles — which could act as building blocks for molecular computer memory and electronic devices (see World’s smallest electronic diode made from single DNA molecule), nanoscale drug-delivery systems, and as markers for biological research and for imaging disease-relevant proteins.

The researchers connected coiled DNA strands between polygon-shaped gold nanoparticles and then reconstructed 3D images, using a cutting-edge electron microscope technique coupled with a protein-staining process and sophisticated software that provided structural details at the scale of about 2 nanometers.

“We had no idea about what the double-strand DNA would look like between the gold nanoparticles,” said Gang “Gary” Ren, a Berkeley Lab scientist who led the research. “This is the first time for directly visualizing an individual double-strand DNA segment in 3D,” he said.

The results were published in an open-access paper in the March 30 edition of Nature Communications.

The method developed by this team, called individual-particle electron tomography (IPET), had earlier captured the 3-D structure of a single protein that plays a key role in human cholesterol metabolism. By grabbing 2D images of an object from different angles, the technique allows researchers to assemble a 3D image of that object.

The team has also used the technique to uncover the fluctuation of another well-known flexible protein, human immunoglobulin 1, which plays a role in the human immune system.

https://youtu.be/lQrbmg9ry90
Berkeley Lab | 3-D Reconstructions of Double strand DNA and Gold Nanoparticle Structures

For this new study of DNA nanostructures, Ren used an electron-beam study technique called cryo-electron microscopy (cryo-EM) to examine frozen DNA-nanogold samples, and used IPET to reconstruct 3-D images from samples stained with heavy metal salts. The team also used molecular simulation tools to test the natural shape variations (“conformations”) in the samples, and compared these simulated shapes with observations.

First visualization of DNA strand dynamics without distorting x-ray crystallography

Ren explained that the naturally flexible dynamics of samples, like a man waving his arms, cannot be fully detailed by any method that uses an average of many observations.

A popular way to view the nanoscale structural details of delicate biological samples is to form them into crystals and zap them with X-rays, but that destroys their natural shape, especially fir the DNA-nanogold samples in this study, which the scientists say are incredibly challenging to crystallize. Other common research techniques may require a collection of thousands of near-identical objects, viewed with an electron microscope, to compile a single, averaged 3-D structure. But an averaged 3D image may not adequately show the natural shape fluctuations of a given object.

The samples in the latest experiment were formed from individual polygon gold nanostructures, measuring about 5 nanometers across, connected to single DNA-segment strands with 84 base pairs. Base pairs are basic chemical building blocks that give DNA its structure. Each individual DNA segment and gold nanoparticle naturally zipped together with a partner to form the double-stranded DNA segment with a gold particle at either end.

https://youtu.be/RDOpgj62PLU
Berkeley Lab | These views compare the various shape fluctuations obtained from different samples of the same type of double-helix DNA segment (DNA renderings in green, 3D reconstructions in purple) connected to gold nanoparticles (yellow).

The samples were flash-frozen to preserve their structure for study with cryo-EM imaging. The distance between the two gold nanoparticles in individual samples varied from 20 to 30 nanometers, based on different shapes observed in the DNA segments.

Researchers used a cryo-electron microscope at Berkeley Lab’s Molecular Foundry for this study. They collected a series of tilted images of the stained objects, and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique.

Sub-nanometer images next

Ren said that the next step will be to work to improve the resolution to the sub-nanometer scale.

“Even in this current state we begin to see 3-D structures at 1- to 2-nanometer resolution,” he said. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami,” Ren said. Researchers hope to build and better characterize nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas in the body.

“DNA is easy to program, synthesize and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” he said. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”

The team included researchers at UC Berkeley, the Kavli Energy NanoSciences Institute at Berkeley Lab and UC Berkeley, and Xi’an Jiaotong University in China. This work was supported by the National Science Foundation, DOE Office of Basic Energy Sciences, National Institutes of Health, the National Natural Science Foundation of China, Xi’an Jiaotong University in China, and the Ministry of Science and Technology in China. View more about Gary Ren’s research group here.


Abstract of Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

 

World’s smallest electronic diode made from single DNA molecule

Electronic components 1,000 times smaller than with silicon may be possible
http://www.kurzweilai.net/worlds-smallest-electronic-diode-made-from-single-dna-molecule
By inserting a small “coralyne” molecule into DNA, scientists were able to create a single-molecule diode (connected here by two gold electrodes), which can be used as an active element in future nanoscale circuits. The diode circuit symbol is shown on the left. (credit: University of Georgia and Ben-Gurion University)

Nanoscale electronic components can be made from single DNA molecules, as researchers at the University of Georgia and at Ben-Gurion University in Israel have demonstrated, using a single molecule of DNA to create the world’s smallest diode.

DNA double helix with base pairs (credit: National Human Genome Research Institute)

A diode is a component vital to electronic devices that allows current to flow in one direction but prevents its flow in the other direction. The development could help stimulate development of DNA components for molecular electronics.

As noted in an open-access Nature Chemistry paper published this week, the researchers designed a 11-base-pair (bp) DNA molecule and inserted a small molecule named coralyne into the DNA.*

They found, surprisingly, that this caused the current flowing through the DNA to be 15 times stronger for negative voltages than for positive voltages, a necessary feature of a diode.

Electronic elements 1,00o times smaller than current components

“Our discovery can lead to progress in the design and construction of nanoscale electronic elements that are at least 1,000 times smaller than current components,” says the study’s lead author, Bingqian Xu an associate professor in the UGA College of Engineering and an adjunct professor in chemistry and physics.

The research team plans to enhance the performance of the molecular diode and construct additional molecular devices, which may include a transistor (similar to a two-layer diode, but with one additional layer).

A theoretical model developed by Yanantan Dubi of Ben-Gurion University indicated the diode-like behavior of DNA originates from the bias voltage-induced breaking of spatial symmetry inside the DNA molecule after the coralyne is inserted.

The research is supported by the National Science Foundation.

*“We prepared the DNA–coralyne complex by specifically intercalating two coralyne molecules into a custom-designed 11-base-pair (bp) DNA molecule (5′-CGCGAAACGCG-3′) containing three mismatched A–A base pairs at the centre,” according to the authors.

UPDATE April 6, 2016 to clarify the coralyne intercalation (insertion) into the DNA molecule.


Abstract of Molecular rectifier composed of DNA with high rectification ratio enabled by intercalation

The predictability, diversity and programmability of DNA make it a leading candidate for the design of functional electronic devices that use single molecules, yet its electron transport properties have not been fully elucidated. This is primarily because of a poor understanding of how the structure of DNA determines its electron transport. Here, we demonstrate a DNA-based molecular rectifier constructed by site-specific intercalation of small molecules (coralyne) into a custom-designed 11-base-pair DNA duplex. Measured current–voltage curves of the DNA–coralyne molecular junction show unexpectedly large rectification with a rectification ratio of about 15 at 1.1 V, a counter-intuitive finding considering the seemingly symmetrical molecular structure of the junction. A non-equilibrium Green’s function-based model—parameterized by density functional theory calculations—revealed that the coralyne-induced spatial asymmetry in the electron state distribution caused the observed rectification. This inherent asymmetry leads to changes in the coupling of the molecular HOMO−1 level to the electrodes when an external voltage is applied, resulting in an asymmetric change in transmission.

 

A stem-cell repair system that can regenerate any kind of human tissue …including disease and aging; human trials next year
http://www.kurzweilai.net/a-stem-cell-repair-system-that-can-regenerate-any-kind-of-human-tissue

http://www.kurzweilai.net/images/spinal_disc_regeneration.jpg

UNSW researchers say the therapy has enormous potential for treating spinal disc injury and joint and muscle degeneration and could also speed up recovery following complex surgeries where bones and joints need to integrate with the body (credit: UNSW TV)

A stem cell therapy system capable of regenerating any human tissue damaged by injury, disease, or aging could be available within a few years, say University of New South Wales (UNSW Australia) researchers.

Their new repair system*, similar to the method used by salamanders to regenerate limbs, could be used to repair everything from spinal discs to bone fractures, and could transform current treatment approaches to regenerative medicine.

The UNSW-led research was published this week in the Proceedings of the National Academy of Sciences journal.

Reprogramming bone and fat cells

The system reprograms bone and fat cells into induced multipotent stem cells (iMS), which can regenerate multiple tissue types and has been successfully demonstrated in mice, according to study lead author, haematologist, and UNSW Associate Professor John Pimanda.

“This technique is a significant advance on many of the current unproven stem cell therapies, which have shown little or no objective evidence they contribute directly to new tissue formation,” Pimanda said. “We have taken bone and fat cells, switched off their memory and converted them into stem cells so they can repair different cell types once they are put back inside the body.”

“We are currently assessing whether adult human fat cells reprogrammed into iMS cells can safely repair damaged tissue in mice, with human trials expected to begin in late 2017.”

http://www.kurzweilai.net/images/UNSW-stem-cell-repair.jpg

Advantages over stem-cell types

There are different types of stem cells including embryonic stem (ES) cells, which during embryonic development generate every type of cell in the human body, and adult stem cells, which are tissue-specific, but don’t regenerate multiple tissue types. Embryonic stem cells cannot be used to treat damaged tissues because of their tumor forming capacity. The other problem when generating stem cells is the requirement to use viruses to transform cells into stem cells, which is clinically unacceptable, the researchers note.

Research shows that up to 20% of spinal implants either don’t heal or there is delayed healing. The rates are higher for smokers, older people and patients with diseases such diabetes or kidney disease.

Human trials are planned next year once the safety and effectiveness of the technique using human cells in mice has been demonstrated.

* The technique involves extracting adult human fat cells and treating them with the compound 5-Azacytidine (AZA), along with platelet-derived growth factor-AB (PDGF-AB) for about two days. The cells are then treated with the growth factor alone for a further two-three weeks.

AZA is known to induce cell plasticity, which is crucial for reprogramming cells. The AZA compound relaxes the hard-wiring of the cell, which is expanded by the growth factor, transforming the bone and fat cells into iMS cells. When the stem cells are inserted into the damaged tissue site, they multiply, promoting growth and healing.

The new technique is similar to salamander limb regeneration, which is also dependent on the plasticity of differentiated cells, which can repair multiple tissue types, depending on which body part needs replacing.

Along with confirming that human adult fat cells reprogrammed into iMS stem cells can safely repair damaged tissue in mice, the researchers said further work is required to establish whether iMS cells remain dormant at the sites of transplantation and retain their capacity to proliferate on demand.

https://youtu.be/zAMCBNujzzw

Abstract of PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

 

First transistors made entirely of nanocrystal ‘inks’ in simplified process

Transistors and other electronic components to be built into flexible or wearable applications; 3D printing planned
http://www.kurzweilai.net/first-transistors-made-entirely-of-nanocrystal-inks
Because this process works at relatively low temperatures, many transistors can be made on a flexible backing at once. (credit: University of Pennsylvania)

University of Pennsylvania engineers have developed a simplified new approach for making transistors by sequentially depositing their components in the form of liquid nanocrystal “inks.” The new process open the door for transistors and other electronic components to be built into flexible or wearable applications. It also avoids the highly complex current process for creating transistors, which requires high-temperature, high-vacuum equipment. Also, the new lower-temperature process is compatible with a wide array of materials and can be applied to larger areas.

Transistors patterned on plastic backing

The researchers’ nanocrystal-based field effect transistors were patterned onto flexible plastic backings using spin coating, but could eventually be constructed by additive manufacturing systems, like 3D printers.

Published in the journal Science,  the study was lead by Cherie Kagan, the Stephen J. Angello Professor in the School of Engineering and Applied Science, and Ji-Hyuk Choi, then a member of her lab, now a senior researcher at the Korea Institute of Geoscience and Mineral Resources. Researchers at Korea University Korea’s Yonsei University were also involved.

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Kagan’s group developed four nanocrystal inks that comprise the transistor, then deposited them on a flexible backing. (credit: University of Pennsylvania)

The researchers began by dispersing a specific type of nanocrystals in a liquid, creating nanocrystal inks. They developed a library of four of these inks: a conductor (silver), an insulator (aluminum oxide), a semiconductor (cadmium selenide), and a conductor combined with a dopant (a mixture of silver and indium). (“Doping” the semiconductor layer of a transistor with impurities controls whether the device creates a positive or negative charge.)

“These materials are colloids just like the ink in your inkjet printer,” Kagan said, “but you can get all the characteristics that you want and expect from the analogous bulk materials, such as whether they’re conductors, semiconductors or insulators.” Although the electrical properties of several of these nanocrystal inks had been independently verified, they had never been combined into full devices. “Our question was whether you could lay them down on a surface in such a way that they work together to form functional transistors.”

Laying down patterns in layers

Such a process entails layering or mixing them in precise patterns.

First, the conductive silver nanocrystal ink was deposited from liquid on a flexible plastic surface that was treated with a photolithographic mask, then rapidly spun to draw it out in an even layer. The mask was then removed to leave the silver ink in the shape of the transistor’s gate electrode.

The researchers followed that layer by spin-coating a layer of the aluminum oxide nanocrystal-based insulator, then a layer of the cadmium selenide nanocrystal-based semiconductor and finally another masked layer for the indium/silver mixture, which forms the transistor’s source and drain electrodes. Upon heating at relatively low temperatures, the indium dopant diffused from those electrodes into the semiconductor component.

“The trick with working with solution-based materials is making sure that, when you add the second layer, it doesn’t wash off the first, and so on,” Kagan said. “We had to treat the surfaces of the nanocrystals, both when they’re first in solution and after they’re deposited, to make sure they have the right electrical properties and that they stick together in the configuration we want.”

Because this entirely ink-based fabrication process works at lower temperatures than existing vacuum-based methods, the researchers were able to make several transistors on the same flexible plastic backing at the same time.

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The inks’ specialized surface chemistry allowed them to stay in configuration without losing their electrical properties. (credit: University of Pennsylvania)

“Making transistors over larger areas and at lower temperatures have been goals for an emerging class of technologies, when people think of the Internet of things, large area flexible electronics and wearable devices,” Kagan said. “We haven’t developed all of the necessary aspects so they could be printed yet, but because these materials are all solution-based, it demonstrates the promise of this materials class and sets the stage for additive manufacturing.”

Because this entirely ink-based fabrication process works at lower temperatures than existing vacuum-based methods, the researchers were able to make several transistors on the same flexible plastic backing at the same time.

3D-printing transistors for wearables

“This is the first work,” Choi said, “showing that all the components, the metallic, insulating, and semiconducting layers of the transistors, and even the doping of the semiconductor, could be made from nanocrystals.”

“Making transistors over larger areas and at lower temperatures have been goals for an emerging class of technologies, when people think of the Internet of things, large area flexible electronics and wearable devices,” Kagan said. “We haven’t developed all of the necessary aspects so they could be printed yet, but because these materials are all solution-based, it demonstrates the promise of this materials class and sets the stage for additive manufacturing.”

The research was supported by the National Science Foundation, the U.S. Department of Energy, the Office of Naval Research, and the Korea Institute of Geoscience and Mineral Resources funded by the Ministry of Science, ICT, and Future Planning of Korea.


Abstract of Exploiting the colloidal nanocrystal library to construct electronic devices

Synthetic methods produce libraries of colloidal nanocrystals with tunable physical properties by tailoring the nanocrystal size, shape, and composition. Here, we exploit colloidal nanocrystal diversity and design the materials, interfaces, and processes to construct all-nanocrystal electronic devices using solution-based processes. Metallic silver and semiconducting cadmium selenide nanocrystals are deposited to form high-conductivity and high-mobility thin-film electrodes and channel layers of field-effect transistors. Insulating aluminum oxide nanocrystals are assembled layer by layer with polyelectrolytes to form high–dielectric constant gate insulator layers for low-voltage device operation. Metallic indium nanocrystals are codispersed with silver nanocrystals to integrate an indium supply in the deposited electrodes that serves to passivate and dope the cadmium selenide nanocrystal channel layer. We fabricate all-nanocrystal field-effect transistors on flexible plastics with electron mobilities of 21.7 square centimeters per volt-second.

Best textile manufacturing methods for creating human tissues with stem cells
Bioengineers determine three best processes for engineering tissues needed for organ and tissue repair
http://www.kurzweilai.net/best-textile-manufacturing-methods-for-creating-human-tissues-with-stem-cells
All four textile manufacturing processes and corresponding scaffold (structure) types studied exhibited the presence of lipid vacuoles (small red spheres, right column, indicating stem cells undergoing random differentiation), compared to control (left). Electrospun scaffolds (row a) exhibited only a monolayer of lipid vacuoles in a single focal plane, while meltblown, spunbond, and carded scaffolds (rows b, c, d) exhibited vacuoles in multiple planes throughout the fabric thickness. Scale bars: 100 μm (credit: S. A. Tuin et al./Biomedical Materials)

Elizabeth Loboa, dean of the Missouri University College of Engineering, and her team have tested new tissue- engineering methods (based on textile manufacturing) to find ones that are most cost-effective and can be produced in larger quantities.

Tissue engineering is a process that uses novel biomaterials seeded with stem cells to grow and replace missing tissues. When certain types of materials are used, the “scaffolds” that are created to hold stem cells eventually degrade, leaving natural tissue in its place. The new tissues could help patients suffering from wounds caused by diabetes and circulation disorders, patients in need of cartilage or bone repair, and women who have had mastectomies by replacing their breast tissue. The challenge is creating enough of the material on a scale that clinicians need to treat patients.

Comparing textile manufacturing techniques

http://www.kurzweilai.net/images/electrospinning.png

Electrospinning experiment: nanofibers are collected into an ethanol bath and removed at predefined time intervals (credit: J. M. Coburn et al./The Johns Hopkins University/PNAS)

In typical tissue engineering approaches that use fibers as scaffolds, non-woven materials are often bonded together using an electrostatic field. This process, called electrospinning (see Nanoscale scaffolds and stem cells show promise in cartilage repair and Improved artificial blood vessels), creates the scaffolds needed to attach to stem cells.

However, large-scale production with electrospinning is not cost-effective. “Electrospinning produces weak fibers, scaffolds that are not consistent, and pores that are too small,” Loboa said. “The goal of ‘scaling up’ is to produce hundreds of meters of material that look the same, have the same properties, and can be used in clinical settings. So we investigated the processes that create textiles, such as clothing and window furnishings like drapery, to scale up the manufacturing process.”

The group published two papers using three industry-standard, high-throughput manufacturing techniques — meltblowing, spunbonding, and carding — to determine if they would create the materials needed to mimic native tissue.

Meltblowing is a technique during which nonwoven materials are created using a molten polymer to create continuous fibers. Spunbond materials are made much the same way but the fibers are drawn into a web while in a solid state instead of a molten one. Carding involves the separation of fibers through the use of rollers, forming the web needed to hold stem cells in place.

http://www.kurzweilai.net/images/carded-scaffold-fabrication.jpg

Schematic of gilled fiber multifilament spinning and carded scaffold fabrication (credit: Stephen A. Tuin et al./Acta Biomaterialia)

Cost-effective methods

Loboa and her colleagues tested these techniques to create polylactic acid (PLA) scaffolds (a Food and Drug Administration-approved material used as collagen fillers), seeded with human stem cells. They then spent three weeks studying whether the stem cells remained healthy and if they began to differentiate into fat and bone pathways, which is the goal of using stem cells in a clinical setting when new bone and/or new fat tissue is needed at a defect site. Results showed that the three textile manufacturing methods proved as viable if not more so than electrospinning.

“These alternative methods are more cost-effective than electrospinning,” Loboa said. “A small sample of electrospun material could cost between $2 to $5. The cost for the three manufacturing methods is between $.30 to $3.00; these methods proved to be effective and efficient. Next steps include testing how the different scaffolds created in the three methods perform once implanted in animals.”

Researchers at North Carolina State University and the University of North Carolina at Chapel Hill were also involved in the two studies, which were published in Biomedical Materials (open access) and Acta Biomaterialia. The National Science Foundation, the National Institutes of Health, and the Nonwovens Institute provided funding for the studies.


Abstract of Creating tissues from textiles: scalable nonwoven manufacturing techniques for fabrication of tissue engineering scaffolds

Electrospun nonwovens have been used extensively for tissue engineering applications due to their inherent similarities with respect to fibre size and morphology to that of native extracellular matrix (ECM). However, fabrication of large scaffold constructs is time consuming, may require harsh organic solvents, and often results in mechanical properties inferior to the tissue being treated. In order to translate nonwoven based tissue engineering scaffold strategies to clinical use, a high throughput, repeatable, scalable, and economic manufacturing process is needed. We suggest that nonwoven industry standard high throughput manufacturing techniques (meltblowing, spunbond, and carding) can meet this need. In this study, meltblown, spunbond and carded poly(lactic acid) (PLA) nonwovens were evaluated as tissue engineering scaffolds using human adipose derived stem cells (hASC) and compared to electrospun nonwovens. Scaffolds were seeded with hASC and viability, proliferation, and differentiation were evaluated over the course of 3 weeks. We found that nonwovens manufactured via these industry standard, commercially relevant manufacturing techniques were capable of supporting hASC attachment, proliferation, and both adipogenic and osteogenic differentiation of hASC, making them promising candidates for commercialization and translation of nonwoven scaffold based tissue engineering strategies.


Abstract of Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications

The fabrication and characterization of novel high surface area hollow gilled fiber tissue engineering scaffolds via industrially relevant, scalable, repeatable, high speed, and economical nonwoven carding technology is described. Scaffolds were validated as tissue engineering scaffolds using human adipose derived stem cells (hASC) exposed to pulsatile fluid flow (PFF). The effects of fiber morphology on the proliferation and viability of hASC, as well as effects of varied magnitudes of shear stress applied via PFF on the expression of the early osteogenic gene marker runt related transcription factor 2 (RUNX2) were evaluated. Gilled fiber scaffolds led to a significant increase in proliferation of hASC after seven days in static culture, and exhibited fewer dead cells compared to pure PLA round fiber controls. Further, hASC-seeded scaffolds exposed to 3 and 6 dyn/cm2 resulted in significantly increased mRNA expression of RUNX2 after one hour of PFF in the absence of soluble osteogenic induction factors. This is the first study to describe a method for the fabrication of high surface area gilled fibers and scaffolds. The scalable manufacturing process and potential fabrication across multiple nonwoven and woven platforms makes them promising candidates for a variety of applications that require high surface area fibrous materials.

Statement of Significance

We report here for the first time the successful fabrication of novel high surface area gilled fiber scaffolds for tissue engineering applications. Gilled fibers led to a significant increase in proliferation of human adipose derived stem cells after one week in culture, and a greater number of viable cells compared to round fiber controls. Further, in the absence of osteogenic induction factors, gilled fibers led to significantly increased mRNA expression of an early marker for osteogenesis after exposure to pulsatile fluid flow. This is the first study to describe gilled fiber fabrication and their potential for tissue engineering applications. The repeatable, industrially scalable, and versatile fabrication process makes them promising candidates for a variety of scaffold-based tissue engineering applications.

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A step forward in diagnostics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions

DARK DAILY   4/8/2016   info@DarkDaily.com  http://www.darkdaily.com/#axzz45En6Xbfr

 

Imaging research is one step closer to giving clinicians a way to do high-resolution scans of malignant cells in order to diagnose cancer and help identify useful therapies. If this technology were to prove successful in clinical studies, it might change how anatomic pathologists and radiologists diagnose and treat cancer.

Researchers at the University of Texas Southwestern Medical Center developed a way to create near-isotropic, high-resolution scans of cells within their microenvironments. The process involves utilizing a combination of two-photonBessel beams and specialized filtering.

New Imaging Approach Could be Useful to Both Pathologists and Radiologists

In a recent press release, senior author Reto Fiolka, PhD, said “there is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned. Our microscope is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor.”

In a study in Developmental Cell, Erik S. Welf, PhD, et al, described the new microenvironmental selective plane illumination microscopy (meSPIM). When developing the technology, the team outlined three goals:

1. The microscope design must not prohibitively constrain microenvironmental properties.

2. Spatial and temporal resolution must match the cellular features of interest.

3. Spatial resolution must be isotropic to avoid spatial bias in quantitative measurements.

This new technology offers pathologists and medical laboratory scientists a new look at cancer cells and other diseases. The study notes that meSPIM eliminates the influence of stiff barriers, such as glass slide covers, while also allowing a level of control over both mechanical and chemical influences that was previously impossible.

Early meSPIM Research Reveals New Cell Behaviors

Early use of meSPIM in observing melanoma cells is already offering new insights into the relationship between the cell behavior of cellular- and subcellular-scale mechanisms and the microenvironment in which these cells exist. The study notes, “The ability to image fine cellular details in controllable microenvironments revealedmorphodynamic features not commonly observed in the narrow range of mechanical environments usually studied in vitro.”

One such difference is the appearance of blebbing. Created by melanoma cells and lines, these small protrusions are thought to aid in cell mobility and survival. Using meSPIM, observers could follow the blebbing process in real-time. Formation of blebs on slides and within an extracellular matrix (ECM) showed significant differences in both formation and manipulation of the surrounding microenvironment.

The team is also using meSPIM to take a look at membrane-associated biosensorand cytosolic biosensor signals in 3D. They hope that investigation of proteins such as phosphatidylinositol 3-kinase (PI3K) and protein kinase C will help to further clarify the roles these signals play in reorientation of fibroblasts.

 

meSPIM-500ppi

meSPIM combined with computer vision enables imaging, visualization, and quantification of how cells alter collagen fibers over large distances within an image volume measuring 100 mm on each side. (Photo Copyright: Welf and Driscoll et al.)   http://www.darkdaily.com/wp-content/uploads/meSPIM-500ppi-220×300.jpg

 

Seeing cancer cells in 3-D (w/ Video)

February 22, 2016

Cancer in 3-D

Extracted surfaces of two cancer cells. (Left) A lung cancer cell colored by actin intensity near the cell surface. Actin is a structural molecule that is integral to cell movement. (Right) A melanoma cell colored by PI3-kinase activity near the cell surface. PI3K is a signaling molecule that is key to many cell processes. Credit: Welf and Driscoll et al.  http://cdn.phys.org/newman/csz/news/800/2016/cancerin3d.png

Cancer cells don’t live on glass slides, yet the vast majority of images related to cancer biology come from the cells being photographed on flat, two-dimensional surfaces—images that are sometimes used to make conclusions about the behaviour of cells that normally reside in a more complex environment. But a new high-resolution microscope, presented February 22 in Developmental Cell, now makes it possible to visualize cancer cells in 3D and record how they are signaling to other parts of their environment, revealing previously unappreciated biology of how cancer cells survive and disperse within living things.

“There is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned,” says senior author Reto Fiolka, an optical scientist at the University of Texas Southwestern Medical Center. “Our is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor environments.”

In their study, Fiolka and colleagues, including co-senior author Gaudenz Danuser, and co-first authors Meghan Driscoll and Erik Welf, also of UT Southwestern, used their microscope to image different kinds of skin cancer cells from patients. They found that in a 3D environment (where cells normally reside), unlike a glass slide, multiple melanoma cell lines and primary melanoma cells (from patients with varied genetic mutations) form many small protrusions called blebs. One hypothesis is that this blebbing may help the survive or move around and could thus play a role in skin cancer cell invasiveness or drug resistance in patients.

The researchers say that this is a first step toward understanding 3D biology in tumor microenvironments. And since these kinds of images may be too complicated to interpret by the naked eye alone, the next step will be to develop powerful computer platforms to extract and process the information.

“When we conceived of this project, we first asked what we wanted to measure and then designed a microscope and analytical platform to achieve this goal,” says co-first author Erik Welf, a cell biologist. “We hope that now instead of asking what we can measure, scientists will ask what we must measure in order to make meaningful contributions to cancer cell biology.”

The microscope control software and image analytical code are freely available to the scientific community.

More information: Developmental Cell, Welf and Driscoll et al.: “Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments” dx.doi.org/10.1016/j.devcel.2016.01.022

Read more at: http://phys.org/news/2016-02-cancer-cells-d-video.html#jCp

Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments

Erik S. Welf4, Meghan K. Driscoll4, Kevin M. Dean, Claudia Schäfer, Jun Chu, Michael W. Davidson, Michael Z. Lin, Gaudenz Danusercorrespondence , Reto Fiolkacorrespondence
Dev Cell  22 Feb 2016;  Volume 36, Issue 4:462–475     DOI: http://dx.doi.org/10.1016/j.devcel.2016.01.022
Highlights
  • meSPIM allows microenvironmentally conscious 3D imaging/analysis of subcellular biology
  • Precisely controlled microenvironments reveal diverse morphological phenotypes
  • Isotropic resolution and high speed enable the quantification of 3D cell signaling and morphodynamics
  • Multiscale quantification of microenvironmental reorganization by cells

Summary

The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments.

Read more: 3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions | Dark Daily http://www.darkdaily.com/3d-imaging-of-cancer-cells-could-lead-to-improved-ability-of-pathologists-and-radiologists-to-plan-cancer-treatments-and-monitor-cell-interactions-301#ixzz45Enp2yT0

The research team believes this opens new possibilities for studying diseases at a subcellular level, saying, “Cell biology is necessarily restricted to studying what we can measure. Accordingly, while the last hundred years have yielded incredible insight into cellular processes, unfortunately most of these studies have involved cells plated onto flat, stiff surfaces that are drastically different from the in vivo microenvironment …

“Here, we introduce an imaging platform that enables detailed subcellular observations without compromising microenvironmental control and thus should open a window for addressing these fundamental questions of cell biology.”

Limitations of meSPIM

One significant issue associated with the use of meSPIM is the need to process the large quantity of data into useful information. Algorithms currently allow for automatic bleb detection. However, manual marking, while time consuming, still provides increased accuracy. Researchers believe the next step in improving the quality of meSPIM scans lie in computer platforms designed to extract and process the scan data.

Until this process is automated, user bias, sample mounting, and data handling will remain risks for introducing errors into the collected data. Yet, even in its early stages, meSPIM offers new options for assessing the state of cancer cells and may eventually provide pathologists and radiologists with additional information when creating treatment plans or assessments.

 

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GE’s $40 Million Center for Additive Technology Advancement (CATA)

UPDATED on 9/6/2016

G.E. Offers $1.4 Billion for 3-D Printing Technology Companies

By CHAD BRAY SEPT. 6, 2016

In GE 3-D printing technology is used to create gas turbine parts and other applications in HealthCare and in Aerospace.

G.E. said it had invested about $1.5 billion in manufacturing and 3-D printing technology since 2010, and added that it expected its new additive manufacturing business to achieve $1 billion in revenue by 2020.

“Additive manufacturing is a key part of G.E.’s evolution into a digital industrial company,” Jeffrey R. Immelt, the G.E. chairman and chief executive, said in a news release.

Arcam, based in Molndal, Sweden, is a provider of metal-based 3-D printing technology, primarily for the aerospace and health care industries. It had $68 million in revenue in 2015 and about 285 employees.

SLM Solutions, based in Lübeck, Germany, which went public two years ago, said in a news release that G.E. had offered to pay 38 euros, about $42.40, a share for the company, a 36.7 percent premium to its closing price on Monday.

If the acquisitions are completed, the companies would report to David L. Joyce, president and chief executive of GE Aviation

SOURCE

GE’s $40 Million Center for Additive Technology Advancement (CATA)

Reporter: Danut Dragoi, PhD

While many are aware of the big names in 3D printing, it still often comes as a surprise to some to find out that General Electric has had their hands in the technology for a long time, and they just keep getting more and more invested. So, if you are wondering about the future of 3D printing or whether or not it’s really catching on, just the fact that GE is opening another multi-million-dollar facility should be a pretty big hint—as well as the fact that they want all of their related businesses getting in on the technology.

It’s also very exciting for us to see what GE is working on further, especially regarding their new Center for Additive Technology Advancement (CATA) in Pittsburgh, which celebrated their grand opening on Tuesday. The city of Pittsburgh is probably most pumped, however, looking forward not just to the activity that the facility will bring, but probably most likely quite happy to have GE declare them as the next industry leader for 3D printing in terms of geography; in fact, the reason GE set up their new $39 million General Electric plant off of a highway exit very near the airport was because of the proximity to Carnegie Mellon University, the University of Pittsburgh and Penn State University—all of whom are very involved in 3D printing—and whose outstanding projects we continue to follow as well. We’ve also followed activity on the part of GE over the years as they have poured millions into 3D printing expansion, and moved into countries like India with multiple facilities.

Now, in the traditional manufacturing setting of Pittsburgh, General Electric is employing numbers of laser 3D printers in the manufacturing of everything from jet engine blades to oil valves. Picture below shows a jet engine blades model that GE engineers produced using an advanced 3-D printing technique called direct metal laser melting. This additive manufacturing method is producing a growing list of parts for numerous industries, making stronger components with less material waste that are impossible to create using traditional techniques.

GE Turbo engine 3DP

Image SOURCE: http://www.ge.com/stories/advanced-manufacturing

CATA is funded by each of the GE businesses, with the goal of integrating 3D printing for all. GE has historically been very involved with 3D printing to create fuel nozzles for jet engines, see picture below.

GE fuel nozle 3DP

Image SOURCE: https://3dprint.com/128490/pittsburgh-ge-cata/

All eight of the company’s manufacturing divisions will use the 125,000-square-foot facility to test new designs and ideas, with 50 high-tech engineers employed there. While currently the CATA facility has just several 3D metal printing machines, they are also, according to GE Reports, going to add an additional $10 million in machines this year, with a $2 million DMLM printer that has four lasers and can print four different components simultaneously.

The CATA facility also holds a sand binder jetting machine, excellent for rapid prototyping. Rather than employing a laser, it uses a chemical binder to use sand as the material for casting molds. Picture below shows a Jell-O mold for the jelly which is a work in progress prototyping for sand binder jetting machine.

Sand binding for molding GE for 3DP

Image SOURCE: https://3dprint.com/128490/pittsburgh-ge-cata/

With their Poly-jet printers, GE engineers are able to combine polymers and make parts with different qualities and colors. The goal is to push the limits of additive manufacturing and stay at the forefront of innovation within the industry. The CATA industrialization lab is meant to promote this mission, allowing GE businesses to bring in their 3D printing concepts and optimize them, as well as working to bring them to fruition. It sounds like they might just be having a little bit of fun in the process too.

Source

https://3dprint.com/128490/pittsburgh-ge-cata/

http://www.ge.com/stories/advanced-manufacturing

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Update on FDA Policy Regarding 3D Bioprinted Material

Curator: Stephen J. Williams, Ph.D.

Last year (2015) in late October the FDA met to finalize a year long process of drafting guidances for bioprinting human tissue and/or medical devices such as orthopedic devices.  This importance of the development of these draft guidances was highlighted in a series of articles below, namely that

  • there were no standards as a manufacturing process
  • use of human tissues and materials could have certain unforseen adverse events associated with the bioprinting process

In the last section of this post a recent presentation by the FDA is given as well as an excellent  pdf here BioprintingGwinnfinal written by a student at University of Kentucky James Gwinn on regulatory concerns of bioprinting.

Bio-Printing Could Be Banned Or Regulated In Two Years

3D Printing News January 30, 2014 No Comments 3dprinterplans

organovaliver

 

 

 

 

 

Cross-section of multi-cellular bioprinted human liver tissue Credit: organovo.com

Bio-printing has been touted as the pinnacle of additive manufacturing and medical science, but what if it might be shut down before it splashes onto the medical scene. Research firm, Gartner Inc believes that the rapid development of bio-printing will spark calls to ban the technology for human and non-human tissue within two years.

A report released by Gartner predicts that the time is drawing near when 3D-bioprinted human organs will be readily available, causing widespread debate. They use an example of 3D printed liver tissue by a San Diego-based company named Organovo.

“At one university, they’re actually using cells from human and non-human organs,” said Pete Basiliere, a Gartner Research Director. “In this example, there was human amniotic fluid, canine smooth muscle cells, and bovine cells all being used. Some may feel those constructs are of concern.”

Bio-printing 

Bio-printing uses extruder needles or inkjet-like printers to lay down rows of living cells. Major challenges still face the technology, such as creating vascular structures to support tissue with oxygen and nutrients. Additionally, creating the connective tissue or scaffolding-like structures to support functional tissue is still a barrier that bio-printing will have to overcome.

Organovo has worked around a number of issues and they hope to print a fully functioning liver for pharmaceutical industry by the end of this year.  “We have achieved thicknesses of greater than 500 microns, and have maintained liver tissue in a fully functional state with native phenotypic behavior for at least 40 days,” said Mike Renard, Organovo’s executive vice president of commercial operations.

clinical trails and testing of organs could take over a decade in the U.S. This is because of the strict rules the U.S. Food and Drug Administration (FDA) places on any new technology. Bio-printing research could outplace regulatory agencies ability to keep up.

“What’s going to happen, in some respects, is the research going on worldwide is outpacing regulatory agencies ability to keep up,” Basiliere said. “3D bio-printing facilities with the ability to print human organs and tissue will advance far faster than general understanding and acceptance of the ramifications of this technology.”

Other companies have been successful with bio-printing as well. Munich-based EnvisionTEC is already selling a printer called a Bioplotter that sells for $188,000 and can print 3D pieces of human tissue. China’s Hangzhou Dianzi University has developed a printer called Regenovo, which printed a small working kidney that lasted four months.

“These initiatives are well-intentioned, but raise a number of questions that remain unanswered. What happens when complex enhanced organs involving nonhuman cells are made? Who will control the ability to produce them? Who will ensure the quality of the resulting organs?” Basiliere said.

Gartner believes demand for bio-printing will explode in 2015, due to a burgeoning population and insufficient levels of healthcare in emerging markets. “The overall success rates of 3D printing use cases in emerging regions will escalate for three main reasons: the increasing ease of access and commoditization of the technology; ROI; and because it simplifies supply chain issues with getting medical devices to these regions,” Basiliere said. “Other primary drivers are a large population base with inadequate access to healthcare in regions often marred by internal conflicts, wars or terrorism.”

It’s interesting to hear Gartner’s bold predictions for bio-printing. Some of the experts we have talked to seem to think bio-printing is further off than many expect, possibly even 20 or 30 years away for fully functioning organs used in transplants on humans. However, less complicated bio-printing procedures and tissue is only a few years away.

 

FDA examining regulations for 3‑D printed medical devices

Renee Eaton Monday, October 27, 2014

fdalogo

The official purpose of a recent FDA-sponsored workshop was “to provide a forum for FDA, medical device manufacturers, additive manufacturing companies and academia to discuss technical challenges and solutions of 3-D printing.” The FDA wants “input to help it determine technical assessments that should be considered for additively manufactured devices to provide a transparent evaluation process for future submissions.”

Simply put, the FDA is trying to stay current with advanced manufacturing technologies that are revolutionizing patient care and, in some cases, democratizing its availability. When a next-door neighbor can print a medical device in his or her basement, it clearly has many positive and negative implications that need to be considered.

Ignoring the regulatory implications for a moment, the presentations at the workshop were fascinating.

STERIS representative Dr. Bill Brodbeck cautioned that the complex designs and materials now being created with additive manufacturing make sterilization practices challenging. For example, how will the manufacturer know if the implant is sterile or if the agent has been adequately removed? Also, some materials and designs cannot tolerate acids, heat or pressure, making sterilization more difficult.

Dr. Thomas Boland from the University of Texas at El Paso shared his team’s work on 3-D-printed tissues. Using inkjet technology, the researchers are evaluating the variables involved in successfully printing skin. Another bio-printing project being undertaken at Wake Forest by Dr. James Yoo involves constructing bladder-shaped prints using bladder cell biopsies and scaffolding.

Dr. Peter Liacouras at Walter Reed discussed his institution’s practice of using 3-D printing to create surgical guides and custom implants. In another biomedical project, work done at Children’s National Hospital by Drs. Axel Krieger and Laura Olivieri involves the physicians using printed cardiac models to “inform clinical decisions,” i.e. evaluate conditions, plan surgeries and reduce operating time.

As interesting as the presentations were, the subsequent discussions were arguably more important. In an attempt to identify and address all significant impacts of additive manufacturing on medical device production, the subject was organized into preprinting (input), printing (process) and post-printing (output) considerations. Panelists and other stakeholders shared their concerns and viewpoints on each topic in an attempt to inform and persuade FDA decision-makers.

An interesting (but expected) outcome was the relative positions of the various stakeholders. Well-established and large manufacturers proposed validation procedures: material testing, process operating guidelines, quality control, traceability programs, etc. Independent makers argued that this approach would impede, if not eliminate, their ability to provide low-cost prosthetic devices.

Comparing practices to the highly regulated food industry, one can understand and accept the need to adopt similar measures for some additively manufactured medical devices. An implant is going into someone’s body, so the manufacturer needs to evaluate and assure the quality of raw materials, processing procedures and finished product.

But, as in the food industry, this means the producer needs to know the composition of materials. Suppliers cannot hide behind proprietary formulations. If manufacturers are expected to certify that a device is safe, they need to know what ingredients are in the materials they are using.

Many in the industry are also lobbying the FDA to agree that manufacturers should be expected to certify the components and not the additive manufacturing process itself. They argue that what matters is whether the device is safe, not what process was used to make it.

Another distinction should be the product’s risk level. Devices should continue to be classified as I, II or III and that classification, not the process used, should determine its level of regulation.

 

 

Will the FDA Regulate Bioprinting?

Published by Sandra Helsel, May 21, 2014 10:20 am

(3DPrintingChannel) The FDA currently assesses 3D printed medical devices and conventionally made products under the same guidelines, despite the different manufacturing methods involved. To receive device approval, manufacturers must prove that the device is equivalent to a product already on the market for the same use, or the device must undergo the process of attaining pre-market approval. However, the approval process for 3D printed devices could become complicated because the devices are manufactured differently and can be customizable. Two teams at the agency are now trying to determine how approval process should be tweaked to account for the changes.

3D Printing and 3D Bioprinting – Will the FDA Regulate Bioprinting?

This entry was posted by Bill Decker on May 20, 2014 at 8:52 am

3dprintedskin

 

 

 

 

 

VIEW VIDEO

https://www.youtube.com/watch?v=5KY-JZCXKXQ#action=share

 

The 3d printing revolution came to medicine and is making people happy while scaring them at the same time!

3-D printing—the process of making a solid object of any shape from a digital model—has grown increasingly common in recent years, allowing doctors to craft customized devices like hearing aids, dental implants, and surgical instruments. For example, University of Michigan researchers last year used a 3-D laser printer to create an airway splint out of plastic particles. In another case, a patient had 75% of his skull replaced with a 3-D printed implant customized to fit his head. The 3d printing revolution came to medicine and is making people happy while scaring them at the same time!

Printed hearts? Doctors are getting there
FDA currently treats assesses 3-D printed medical devices and conventionally made products under the same guidelines, despite the different manufacturing methods involved. To receive device approval, manufacturers must prove that the device is equivalent to a product already on the market for the same use, or the device must undergo the process of attaining pre-market approval.

“We evaluate all devices, including any that utilize 3-D printing technology, for safety and effectiveness, and appropriate benefit and risk determination, regardless of the manufacturing technologies used,” FDA spokesperson Susan Laine said.
However, the approval process for 3-D printed devices could become complicated because the devices are manufactured differently and can be customizable. Two teams at the agency now are trying to determine how approval process should be tweaked to account for the changes:

http://product-liability.weil.com/news/the-stuff-of-innovation-3d-bioprinting-and-fdas-possible-reorganization/

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The Stuff of Innovation – 3D Bioprinting and FDA’s Possible Reorganization

Weil Product Liability Monitor on September 10, 2013 ·

Posted in News

Contributing Author: Meghan A. McCaffrey

With 3D printers, what used to exist only in the realm of science fiction — who doesn’t remember the Star Trek food replicator that could materialize a drink or meal with the mere press of a button — is now becoming more widely available with  food on demand, prosthetic devices, tracheal splintsskull implants, and even liver tissue all having recently been printed, used, implanted or consumed.  3D printing, while exciting, also presents a unique hybrid of technology and biology, making it a potentially unique and difficult area to regulate and oversee.  With all of the recent technological advances surround 3D printer technology, the FDA recently announced in a blog post that it too was going 3D, using it to “expand our research efforts and expand our capabilities to review innovative medical products.”  In addition, the agency will be investigating how 3D printing technology impacts medical devices and manufacturing processes.  This will, in turn, raise the additional question of how such technology — one of the goals of which, at least in the medical world,  is to create unique and custom printed devices, tissue and other living organs for use in medical procedures — can be properly evaluated, regulated and monitored.
In medicine, 3D printing is known as “bioprinting,” where so-called bioprinters print cells in liquid or gel format in an attempt to engineer cartilage, bone, skin, blood vessels, and even small pieces of liver and other human tissues [see a recent New York Times article here].  Not to overstate the obvious, but this is truly cutting edge science that could have significant health and safety ramifications for end users.  And more importantly for regulatory purposes, such bioprinting does not fit within the traditional category of a “device” or a “biologic.”  As was noted in Forbes, “more of the products that FDA is tasked with regulating don’t fit into the traditional categories in which FDA has historically divided its work.  Many new medical products transcend boundaries between drugs, devices, and biologics…In such a world, the boundaries between FDA’s different centers may no longer make as much sense.”  To that end, Forbes reported that FDA Commissioner Peggy Hamburg announced Friday the formation of a “Program Alignment Group” at the FDA whose goal is to identify and develop plans “to best adapt to the ongoing rapid changes in the regulatory environment, driven by scientific innovation, globalization, the increasing complexity of regulated products, new legal authorities and additional user fee programs.”

It will be interesting to see if the FDA can retool the agency to make it a more flexible, responsive, and function-specific organization.  In the short term, the FDA has tasked two laboratories in the Office of Science and Engineering Laboratories with investigating how the new 3D technology can impact the safety and efficacy of devices and materials manufactured using the technology.  The Functional Performance and Device Use Laboratory is evaluating “the effect of design changes on the safety and performance of devices when used in different patient populations” while the Laboratory for Solid Mechanics is assessing “how different printing techniques and processes affect the strength and durability of the materials used in medical devices.”  Presumably, all of this information will help the FDA evaluate at some point in the future whether a 3D printed heart is safe and effective for use in the patient population.

In any case, this type of hybrid technology can present a risk for companies and manufacturers creating and using such devices.  It remains to be seen what sort of regulations will be put in place to determine, for example, what types of clinical trials and information will have to be provided before a 3D printer capable of printing a human heart is approved for use by the FDA.  Or even on a different scale, what regulatory hurdles (and on-going monitoring, reporting, and studies) will be required before bioprinted cartilage can be implanted in a patient’s knee.  Are food replicators and holodecks far behind?

http://www.raps.org/regulatory-focus/news/2014/05/19000/FDA-3D-Printing-Guidance-and-Meeting/

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FDA Plans Meeting to Explore Regulation, Medical Uses of 3D Printing Technology

Posted 16 May 2014 By Alexander Gaffney, RAC

The US Food and Drug Administration (FDA) plans to soon hold a meeting to discuss the future of regulating medical products made using 3D printing techniques, it has announced.

fdaplanstomeetbioprinting

Background

3D printing is a manufacturing process which layers printed materials on top of one another, creating three-dimensional parts (as opposed to injection molding or routing materials).

The manufacturing method has recently come into vogue with hobbyists, who have been driven by several factors only likely to accelerate in the near future:

  • The cost of 3D printers has come down considerably.
  • Electronic files which automate the printing process are shareable over the Internet, allowing anyone with the sufficient raw materials to build a part.
  • The technology behind 3D printing is becoming more advanced, allowing for the manufacture of increasingly durable parts.

While the technology has some alarming components—the manufacture of untraceable weapons, for example—it’s increasingly being looked at as the future source of medical product innovation, and in particular for medical devices like prosthetics.

Promise and Problems

But while 3D printing holds promise for patients, it poses immense challenges for regulators, who must assess how to—or whether to—regulate the burgeoning sector.

In a recent FDA Voice blog posting, FDA regulators noted that 3D-printed medical devices have already been used in FDA-cleared clinical interventions, and that it expects more devices to emerge in the future.

Already, FDA’s Office of Science and Engineering laboratories are working to investigate how the technology will affect the future of device manufacturing, and CDRH’s Functional Performance and Device Use Laboratory is developing and adapting computer modeling methods to help determine how small design changes could affect the safety of a device. And at the Laboratory for Solid Mechanics, FDA said it is investigating the materials used in the printing process and how those might affect durability and strength of building materials.

And as Focus noted in August 2013, there are myriad regulatory challenges to confront as well. For example: If a 3D printer makes a medical device, will that device be considered adulterated since it was not manufactured under Quality System Regulation-compliant conditions? Would each device be required to be registered with FDA? And would FDA treat shared design files as unauthorized promotion if they failed to make proper note of the device’s benefits and risks? What happens if a device was never cleared or approved by FDA?

The difficulties for FDA are seemingly endless.

Plans for a Guidance Document

But there have been indications that FDA has been thinking about this issue extensively.

In September 2013, Focus first reported that CDRH Director Jeffery Shuren was planning to release a guidance on 3D printing in “less than two years.”

Responding to Focus, Shuren said the guidance would be primarily focused on the “manufacturing side,” and probably on how 3D printing occurs and the materials used rather than some of the loftier questions posed above.

“What you’re making, and how you’re making it, may have implications for how safe and effective that device is,” he said, explaining how various methods of building materials can lead to various weaknesses or problems.

“Those are the kinds of things we’re working through. ‘What are the considerations to take into account?'”

“We’re not looking to get in the way of 3D printing,” Shuren continued, noting the parallel between 3D printing and personalized medicine. “We’d love to see that.”

Guidance Coming ‘Soon’

In recent weeks there have been indications that the guidance could soon see a public release. Plastics News reported that CDRH’s Benita Dair, deputy director of the Division of Chemistry and Materials Science, said the 3D printing guidance would be announced “soon.”

“In terms of 3-D printing, I think we will soon put out a communication to the public about FDA’s thoughts,” Dair said, according to Plastics News. “We hope to help the market bring new devices to patients and bring them to the United States first. And we hope to play an integral part in that.”

Public Meeting

But FDA has now announced that it may be awaiting public input before it puts out that guidance document. In a 16 May 2014 Federal Register announcement, the agency said it will hold a meeting in October 2014 on the “technical considerations of 3D printing.”

“The purpose of this workshop is to provide a forum for FDA, medical device manufacturers, additive manufacturing companies, and academia to discuss technical challenges and solutions of 3-D printing. The Agency would like input regarding technical assessments that should be considered for additively manufactured devices to provide a transparent evaluation process for future submissions.”

That language—”transparent evaluation process for future submissions”—indicates that at least one level, FDA plans to treat 3D printing no differently than any other medical device, subjecting the products to the same rigorous premarket assessments that many devices now undergo.

FDA’s notice seems to focus on industrial applications for the technology—not individual ones. The agency notes that it has already “begun to receive submissions using additive manufacturing for both traditional and patient-matched devices,” and says it sees “many more on the horizon.”

Among FDA’s chief concerns, it said, are process verification and validation, which are both key parts of the medical device quality manufacturing regulations.

But the notice also indicates that existing guidance documents, such as those specific to medical device types, will still be in effect regardless of the 3D printing guidance.

Discussion Points

FDA’s proposed list of discussion topics include:

  • Preprinting considerations, including but not limited to:
    • material chemistry
    • physical properties
    • recyclability
    • part reproducibility
    • process validation
  • Printing considerations, including but not limited to:
    • printing process characterization
    • software used in the process
    • post-processing steps (hot isostatic pressing, curing)
    • additional machining
  • Post-printing considerations, including but not limited to:
    • cleaning/excess material removal
    • effect of complexity on sterilization and biocompatibility
    • final device mechanics
    • design envelope
    • verification

– See more at: http://www.raps.org/regulatory-focus/news/2014/05/19000/FDA-3D-Printing-Guidance-and-Meeting/#sthash.cDg4Utln.dpuf

 

FDA examining regulations for 3‑D printed medical devices

 

Renee Eaton Monday, October 27, 2014

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The official purpose of a recent FDA-sponsored workshop was “to provide a forum for FDA, medical device manufacturers, additive manufacturing companies and academia to discuss technical challenges and solutions of 3-D printing.” The FDA wants “input to help it determine technical assessments that should be considered for additively manufactured devices to provide a transparent evaluation process for future submissions.”

Simply put, the FDA is trying to stay current with advanced manufacturing technologies that are revolutionizing patient care and, in some cases, democratizing its availability. When a next-door neighbor can print a medical device in his or her basement, it clearly has many positive and negative implications that need to be considered.

Ignoring the regulatory implications for a moment, the presentations at the workshop were fascinating.

STERIS representative Dr. Bill Brodbeck cautioned that the complex designs and materials now being created with additive manufacturing make sterilization practices challenging. For example, how will the manufacturer know if the implant is sterile or if the agent has been adequately removed? Also, some materials and designs cannot tolerate acids, heat or pressure, making sterilization more difficult.

Dr. Thomas Boland from the University of Texas at El Paso shared his team’s work on 3-D-printed tissues. Using inkjet technology, the researchers are evaluating the variables involved in successfully printing skin. Another bio-printing project being undertaken at Wake Forest by Dr. James Yoo involves constructing bladder-shaped prints using bladder cell biopsies and scaffolding.

Dr. Peter Liacouras at Walter Reed discussed his institution’s practice of using 3-D printing to create surgical guides and custom implants. In another biomedical project, work done at Children’s National Hospital by Drs. Axel Krieger and Laura Olivieri involves the physicians using printed cardiac models to “inform clinical decisions,” i.e. evaluate conditions, plan surgeries and reduce operating time.

As interesting as the presentations were, the subsequent discussions were arguably more important. In an attempt to identify and address all significant impacts of additive manufacturing on medical device production, the subject was organized into preprinting (input), printing (process) and post-printing (output) considerations. Panelists and other stakeholders shared their concerns and viewpoints on each topic in an attempt to inform and persuade FDA decision-makers.

An interesting (but expected) outcome was the relative positions of the various stakeholders. Well-established and large manufacturers proposed validation procedures: material testing, process operating guidelines, quality control, traceability programs, etc. Independent makers argued that this approach would impede, if not eliminate, their ability to provide low-cost prosthetic devices.

Comparing practices to the highly regulated food industry, one can understand and accept the need to adopt similar measures for some additively manufactured medical devices. An implant is going into someone’s body, so the manufacturer needs to evaluate and assure the quality of raw materials, processing procedures and finished product.

But, as in the food industry, this means the producer needs to know the composition of materials. Suppliers cannot hide behind proprietary formulations. If manufacturers are expected to certify that a device is safe, they need to know what ingredients are in the materials they are using.

Many in the industry are also lobbying the FDA to agree that manufacturers should be expected to certify the components and not the additive manufacturing process itself. They argue that what matters is whether the device is safe, not what process was used to make it.

Another distinction should be the product’s risk level. Devices should continue to be classified as I, II or III and that classification, not the process used, should determine its level of regulation.

If you are interested in submitting comments to the FDA on this topic, post them by Nov. 10.

FDA Guidance Summary on 3D BioPrinting

fdaregulationguidelinesfor3dbioprinting_1 fdaregulationguidelinesfor3dbioprinting_2 fdaregulationguidelinesfor3dbioprinting_3 fdaregulationguidelinesfor3dbioprinting_4 fdaregulationguidelinesfor3dbioprinting_5 fdaregulationguidelinesfor3dbioprinting_6 fdaregulationguidelinesfor3dbioprinting_7 fdaregulationguidelinesfor3dbioprinting_8 fdaregulationguidelinesfor3dbioprinting_9 fdaregulationguidelinesfor3dbioprinting_10 fdaregulationguidelinesfor3dbioprinting_11

 

 

 

 

 

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Bio-inks and 3D BioPrinting

Curator: Stephen J. Williams, Ph.D.

 

Bio-ink is a material made from living cells that behaves much like a liquid, allowing people to “print” it in order to create a desired shape. This material was developed by researchers at the University of Missouri, Columbia, with the goal of someday being able to do things like print replacements for failing organs. This technology is only in the very early stages of testing and development, but it shows promise.

To make bio-ink, scientists create a slurry of cells that can be loaded into a cartridge and inserted into a specially designed printer, along with another cartridge containing a gel known as bio-paper. After inputting the standards for the thing they want to print, the researchers trigger the printer, and the cartridges alternate layers to build a three dimensional structure, with the bio-paper creating a supportive matrix that the ink can thrive on.

Through a process that is not yet totally understood, the individual droplets fuse together, eventually latticing upwards through the bio-paper to create a solid structure. Understanding this process and the point at which cells differentiate to accomplish different tasks is an important part of creating a usable material; perhaps someday hospitals will be able to use it to generate tissue and organs for use by their patients.

 

The most obvious potential use for bio-ink is in skin grafting. With this technology, labs could quickly create sheets of skin for burn victims and other people who might be in need of grafts. By creating grafts derived from the patient’s own cells, it could reduce the risk of rejection and scarring. Bio-ink could also be used to make replacements for vascular material removed during surgeries, allowing people to receive new veins and arteries.

Eventually, entire organs could be constructed from this material. Since organs are in short supply around the world, bio-ink could potentially save untold numbers of lives, as patients would no longer have to wait on the transplant list for new organs. The use of such organs could also allay fears about contaminated organ supplies or unscrupulous organ acquisition methods.

 

RegenHu

Universal Matrix for 3D Tissue Printing

BioInkTM is a chemically-defined hydrogel to support growth of different cell types. It allows cell adhesion, mimics the natural extracellular matrix and is biodegradable.

BioInkTM is provided as a ready-to-use chemically-defined hydrogel to print 3D tissue models. Exclusively designed for regenHU’s BioFactory® and 3DDiscovery® tissue and bio-printers.

A versatile, chemically-defined hydrogel, supporting cell attachment, growth, differentiation and migration. The BioInkTM is suitable for long-term tissue cultivation (in vitro human dermis for up to 7 weeks).

 

 

 

 

 

 

 

A versatile bioink for three-dimensional printing of cellular scaffolds based on thermally and photo-triggered tandem gelation

  • a Cartilage Engineering + Regeneration Laboratory, ETH Zürich, Otto-Stern-Weg 7, 8093 Zürich, Switzerland
  • b Biomaterials Department, INNOVENT e.V. Jena, Prüssingstrasse 27 B, 07745 Jena, Germany
  • c AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos Platz, Switzerland

 

Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while HAMA ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.

bioink presentation_1 bioink presentation_2 bioink presentation_3 bioink presentation_4 bioink presentation_5 bioink presentation_6 bioink presentation_7 bioink presentation_8 bioink presentation_9 bioink presentation_10 bioink presentation_11 bioink presentation_12 bioink presentation_13 bioink presentation_14 bioink presentation_15

 

https://www.youtube.com/watch?v=9D749wZSlb0

For more information see:

http://www.slideshare.net/StephenJWilliamsPhD/clipboards/my-clips

 

And for more information on biopaper and methodology please see this pdf file courtesy of The First Symposium on BioPrinting in Tissue Engineering (see file) biopaper presentation

 

 

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Augmented Meta-reality

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

TED releases Meta 2 augmented-reality presentation video

http://www.kurzweilai.net/ted-releases-meta-2-augmented-reality-presentation-video

TED just released the full video of Meta CEO Meron Gribetz’s preview of Meta’s next-generation augmented reality (AR) technology at the TED 2016 conference on Feb. 17. It can be found online at metavision.com and TED.com.

The presentation, which Forbes said “dazzles TED crowd” and received a standing ovation from TED attendees, dramatically showcases the capabilities of the Meta 2 Development Kit. Launched two weeks ago, the Meta 2 kit is now available for pre-order at $949 at metavision.com. (Also see “First ‘natural machine’ augmented reality product Meta 2 launches to developers.)

http://www.ted.com/talks/meron_gribetz_a_glimpse_of_the_future_through_an_augmented_reality_headset#

 

comments

According to available information, Meta devices use holographic imagery for human-machine interfaces. HoloTouch, Inc. has developed significant patented technology in the field of human-machine interfaces by means of holographic imagery. See http://holotouch.com   McPheters

The direct link to the video can be found athttp://www.ted.com/talks/quick-list for those who (like Google) have issues with “flash”.

I think that the most interesting point Meron made was about shared interactions, that will be the “killer app” for AR.

I did notice one thing that may cause Meron problems if he wants to be using AR all the time, he talks with his hands as much as I do. 🙂

AR systems will need to be able to differentiate between command gestures and language gestures. Without this ability AR could be dangerous to use in many contexts. The simplest way would to not act on gestures when the user is talking, but this limitation may be unreasonable. Only an intelligent, adaptive, system could be sophisticated enough at interpreting clues to do this, therefore the future success of AR depends on AI.

Keep in mind that it is not just an issue of the user having self control, because we may also have involuntary movements to, e.g. sudden loud noises, or the use of taboo words such as “nigger”, depending on our reflexes and our social conditioning. You don’t want to have a nasty accident because some sociopath trolled you.    DSM

Alternatively, one may await this: https://www.youtube.com/watch?v=AoWi10YVmfE   JordanMicahBennett

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Real Time 3 D Holograms

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Next-Gen Holographic Microscope Offers Real-Time 3D Imaging

http://www.rdmag.com/news/2016/03/next-gen-holographic-microscope-offers-real-time-3d-imaging       KAIST

3-D Images of representative biological cells taken with the HT-1

3-D Images of representative biological cells taken with the HT-1

Researchers have developed a powerful method for 3D imaging of live cells without staining.

Professor YongKeun Park of the Physics Department at the Korea Advanced Institute of Science and Technology (KAIST) is a leading researcher in the field of biophotonics and has dedicated much of his research career to working on digital holographic microscopy technology. Park and his research team collaborated with the R&D team of a start-up that Park co-founded to develop a state-of-the-art, 2D/3D/4D holographic microscope that would allow a real-time label-free visualization of biological cells and tissues.

The HT is an optical analogy of X-ray computed tomography (CT). Both X-ray CT and HT share the same physical principle—the inverse of wave scattering. The difference is that HT uses laser illumination, whereas X-ray CT uses X-ray beams. From the measurement of multiple 2D holograms of a cell, coupled with various angles of laser illuminations, the 3-D refractive index (RI) distribution of the cell can be reconstructed. The reconstructed 3D RI map provides structural and chemical information of the cell including mass, morphology, protein concentration and dynamics of the cellular membrane.

The HT enables users to quantitatively and non-invasively investigate the intrinsic properties of biological cells, for example, dry mass and protein concentration. Some of the research team’s breakthroughs that have leveraged HT’s unique and special capabilities can be found in several recent publications, including a lead article on the simultaneous 3-D visualization and position tracking of optically trapped particles which was published in Optica on April 20, 2015.

Current fluorescence confocal microscopy techniques require the use of exogenous labeling agents to render high-contrast molecular information. Therefore, drawbacks include possible

  • photo-bleaching
  • photo-toxicity
  • interference with normal molecular activities

Immune or stem cells that need to be reinjected into the body are considered particularly difficult to employ with fluorescence microscopy.

“As one of the two currently available, high-resolution tomographic microscopes in the world, I believe that the HT-1 is the best-in-class regarding specifications and functionality. Users can see 3D/4D live images of cells, without fixing, coating or staining cells. Sample preparation times are reduced from a few days or hours to just a few minutes,” said Park.

“Our technology has set a new paradigm for cell observation under a microscope. I expect that this tomographic microscopy will be more widely used in future in various areas of pharmaceuticals, neuroscience, immunology, hematology and cell biology,” Park added.

The researchers announced the launch of their new microscopic tool, the holotomography (HT)-1, to the global marketplace through the Korean start-up TomoCube. Two Korean hospitals, Seoul National University Hospital in Bundang and Boramae Hospital in Seoul, are currently using this microscope. The research team has also introduced the HT-1 at the Photonics West Exhibition 2016 that took place on February 16-18, 2016, in San Francisco, CA.

 

Chip-Based Atomic Physics Makes Second Quantum Revolution a Reality

http://www.rdmag.com/news/2016/03/chip-based-atomic-physics-makes-second-quantum-revolution-reality

A quartz surface above the electrodes used to trap atoms. The color map on the surface shows the electric field amplitude.

A quartz surface above the electrodes used to trap atoms. The color map on the surface shows the electric field amplitude.

A University of Oklahoma-led team of physicists believes chip-based atomic physics holds promise to make the second quantum revolution—the engineering of quantum matter with arbitrary precision—a reality. With recent technological advances in fabrication and trapping, hybrid quantum systems are emerging as ideal platforms for a diverse range of studies in quantum control, quantum simulation and computing.

James P. Shaffer, professor in the Homer L. Dodge Department of Physics and Astronomy, OU College of Arts and Sciences; Jon Sedlacek, OU graduate student; and a team from the University of Nevada, Western Washington University, The United States Naval Academy, Sandia National Laboratories and Harvard-Smithsonian Center for Astrophysics, have published research important for integrating Rydberg atoms into hybrid quantum systems and the fundamental study of atom-surface interactions, as well as applications for electrons bound to a 2-D surface.

“A convenient surface for application in hybrid quantum systems is quartz because of its extensive use in the semiconductor and optics industries,” Sedlacek said. “The surface has been the subject of recent interest as a result of it stability and low surface energy. Mitigating electric fields near ‘trapping’ surfaces is the holy grail for realizing hybrid quantum systems,” added Hossein Sadeghpour, director of the Institute for Theoretical Atomic Molecular and Optical Physics, Harvard-Smithsonian Center for Astrophysics.

In this work, Shaffer finds ionized electrons from Rydberg atoms excited near the quartz surface form a 2-D layer of electrons above the surface, canceling the electric field produced by rubidium surface adsorbates. The system is similar to electron trapping in a 2-D gas on superfluid liquid helium. The binding of electrons to the surface substantially reduces the electric field above the surface.

“Our results show that binding is due to the image potential of the electron inside the quartz,” said Shaffer. “The electron can’t diffuse into the quartz because the rubidium adsorbates make the surface have a negative electron affinity. The approach is a promising pathway for coupling Rydberg atoms to surfaces, as well as for using surfaces close to atomic and ionic samples.”

A paper on this research was published in the American Physics Society’s Physical Review Letters. The OU part of this work was supported by the Defense Advanced Research Projects Agency Quasar program by a grant through the Army Research Office, the Air Force Office of Scientific Research and the National Science Foundation.

 

New Spin on Biomolecular Tags Lets MRI Catch Metabolic Wobbles

http://www.genengnews.com/gen-news-highlights/new-spin-on-biomolecular-tags-lets-mri-catch-metabolic-wobbles/81252531/

Duke scientists have discovered a new class of inexpensive and long-lived molecular tags that enhance MRI signals by 10,000-fold. To activate the tags, the researchers mix them with a newly developed catalyst (center) and a special form of hydrogen (gray), converting them into long-lived magnetic resonance “lightbulbs” that might be used to track disease metabolism in real time. [Thomas Theis, Duke University]

In principle, magnetic resonance imaging (MRI) could be used to track disease-related biomolecular processes. In practice, magnetic resonance signals die out too quickly. Also, these signals are detectable only with incredibly expensive equipment. The necessary devices, called hyperpolarizers, are commercially available, but they cost as much as $3 million each.

Yet magnetic resonance can be more practical, report scientists from Duke University. These scientists say that they have discovered a new class of molecular tags that enhance magnetic resonance signals by 10,000-fold and generate detectable signals that last over an hour, and not just a few seconds, as is the case with currently available tags. Moreover, the tags are biocompatible and inexpensive to produce, paving the way for widespread use of MRI to monitor metabolic process of conditions such as cancer and heart disease in real time.

According to the Duke team, which was led by physicist Warren S. Warren, Ph.D., and chemist Thomas Theis, Ph.D., the hyperpolarization window to in vitro and in vivo biochemistry can be opened by combining two advances: (1) the use of 15N2-diazirines as storage vessels for hyperpolarization, and (2) a relatively simple and inexpensive approach to hyperpolarization called SABRE-SHEATH.

The details appeared March 25 in the journal Science Advances, in an article entitled, “Direct and Cost-Efficient Hyperpolarization of Long-Lived Nuclear Spin States on Universal 15N2-Diazirine Molecular Tags.” The article explains that the promise of magnetic resonance in tracking chemical transformations has not been realized because of the limitations of existing techniques, such as dissolution dynamic nuclear polarization (d-DNP). Such techniques have lacked adequate sensitivity and are unable to detect small number of molecules without using unattainably massive magnetic fields.

MRI takes advantage of a property called spin, which makes the nuclei in hydrogen atoms act like tiny magnets. Applying a strong magnetic field, followed by a series of radio waves, induces these hydrogen magnets to broadcast their locations. Most of the hydrogen atoms in the body are bound up in water; therefore, the technique is used in clinical settings to create detailed images of soft tissues like organs, blood vessels, and tumors inside the body.

With greater sensitivity, however, magnetic resonance techniques could be used to track chemical transformations in real time. This degree of sensitivity, say the Duke scientists, could be within reach.

“We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize 15N2 magnetization and long-lived 15N2 singlet spin order, with signal decay time constants of 5.8 and 23 minutes, respectively,” wrote the authors of the Science Advances article. “We find >10,000-fold enhancements generating detectable nuclear MR signals that last for over an hour.” The authors added that 15N2-diazirines represent a class of particularly promising and versatile molecular tags and can be incorporated into a wide range of biomolecules without significantly altering molecular function.

“This represents a completely new class of molecules that doesn’t look anything at all like what people thought could be made into MRI tags,” said Dr. Warren “We envision it could provide a whole new way to use MRI to learn about the biochemistry of disease.”

Qiu Wang, Ph.D., an assistant professor of chemistry at Duke and co-author on the paper, said the structure of 15N2-diazirine is a particularly exciting target for hyperpolarization because it has already been demonstrated as a tag for other types of biomedical imaging.

“It can be tagged on small molecules, macromolecules, amino acids, without changing the intrinsic properties of the original compound,” said Dr. Wang. “We are really interested to see if it would be possible to use it as a general imaging tag.” Magnetic resonance, added Dr. Theis, is uniquely sensitive to chemical transformations: “With magnetic resonance, you can see and track chemical transformations in real time.”

The scientists believe their SABRE-SHEATH catalyst could be used to hyperpolarize a wide variety of chemical structures at a fraction of the cost of other methods. “You could envision, in five or ten years, you’ve got the container with the catalyst, you’ve got the bulb with the hydrogen gas,” explained Dr. Warren. “In a minute, you’ve made the hyperpolarized agent, and on the fly you could actually take an image. That is something that is simply inconceivable by any other method.”

 

Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal 15N2-diazirine molecular tags
Conventional magnetic resonance (MR) faces serious sensitivity limitations which can be overcome by hyperpolarization methods, but the most common method (dynamic nuclear polarization) is complex and expensive, and applications are limited by short spin lifetimes (typically seconds) of biologically relevant molecules. We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize 15N2 magnetization and long-lived 15N2 singlet spin order, with signal decay time constants of 5.8 and 23 minutes, respectively. We find >10,000-fold enhancements generating detectable nuclear MR signals that last for over an hour. 15N2-diazirines represent a class of particularly promising and versatile molecular tags, and can be incorporated into a wide range of biomolecules without significantly altering molecular function.

Hyperpolarization enables real-time monitoring of in vitro and in vivo biochemistry

Conventional magnetic resonance (MR) is an unmatched tool for determining molecular structures and monitoring structural transformations. However, even very large magnetic fields only slightly magnetize samples at room temperature and sensitivity remains a fundamental challenge; for example, virtually all MR images are of water because it is the molecule at the highest concentration in vivo. Nuclear spin hyperpolarization significantly alters this perspective by boosting nuclear MR (NMR) sensitivity by four to nine orders of magnitude (13), giving access to detailed chemical information at low concentrations. These advances are beginning to transform biomedical in vivo applications (49) and structural in vitro studies (1016).

Current hyperpolarization technology is expensive and associated with short signal lifetimes

Still, two important challenges remain. First, hyperpolarized MR is associated with high cost for the most widespread hyperpolarization technology [dissolution dynamic nuclear polarization (d-DNP), $2 million to $3 million for commercial hyperpolarizers]. Second, hyperpolarized markers typically have short signal lifetimes: typically, hyperpolarized signals may only be tracked for 1 to 2 min in the most favorable cases (6), greatly limiting this method as a probe for slower biological processes.

The presented approach is inexpensive and produces long-lived signals

Here, we demonstrate that both of these challenges can be overcome simultaneously, setting the stage for hour-long tracking of molecular markers with inexpensive equipment. Specifically, we illustrate the potential of 15N2-diazirines as uniquely powerful storage vessels for hyperpolarization. We show that diazirine can be hyperpolarized efficiently and rapidly (literally orders of magnitude cheaper and quicker than d-DNP), and that this hyperpolarization can be induced in states that maintain hyperpolarization for more than an hour.

Our approach uses parahydrogen (p-H2) to directly polarize long-lived nuclear spin states. The first demonstration of parahydrogen-induced polarization (PHIP) was performed in the late 1980s (1719). Then, PHIP was used to rely on the addition of p-H2 to a carbon double or triple bond, incorporating highly polarized hydrogen atoms into molecules. This approach generally requires specific catalyst-substrate pairs; in addition, hydrogen atoms usually have short relaxation times (T1) that cause signal decay within a few seconds. A more recent variant, SABRE (signal amplification by reversible exchange) (20, 21), uses p-H2 to polarize 1H atoms on a substrate without hydrogenation. In SABRE, both p-H2 and substrate reversibly bind to an iridium catalyst and the hyperpolarization is transferred from p-H2 to the substrate through J-couplings established on the catalytic intermediate. Recently, we extended this method to SABRE-SHEATH (SABRE in SHield Enables Alignment Transfer to Heteronuclei) for direct hyperpolarization of 15N molecular sites (2224). This method has several notable features. Low-γ nuclei (13C, 15N) tend to have long relaxation times, particularly if a proton is not attached. In addition, conventional SABRE relies on small differences between four-bond proton-proton J-couplings (detailed in the Supplementary Materials), whereas SABRE-SHEATH uses larger two-bond heteronuclear J-couplings. It is extremely simple: SABRE-SHEATH requires nothing but p-H2, the catalyst, and a shield to reduce Earth’s field by about 99%. After 1 to 5 min of bubbling p-H2into the sample in the shield, we commonly achieve 10% nitrogen polarization, many thousands of times stronger than thermal signals (22). In contrast, d-DNP typically produces such polarization levels in an hour, at much higher cost.

Diazirines are small and versatile molecular tags

A general strategy for many types of molecular imaging is the creation of molecular tags, which ideally do not alter biochemical pathways but provide background-free signatures for localization. This strategy has not been very successful in MR because of sensitivity issues. Here, we demonstrate that SABRE-SHEATH enables a MR molecular beacon strategy using diazirines Embedded Image (three-membered rings containing a nitrogen-nitrogen double bond). They are highly attractive as molecular tags, primarily because of their small size. Diazirines have already been established as biocompatible molecular tags for photoaffinity labeling (25). They can be incorporated into many small molecules, metabolites, and biomolecules without drastically altering biological function. Diazirines share similarities with methylene (CH2) groups in terms of electronic and steric properties such that they can replace methylene groups without drastically distorting biochemical behavior. Furthermore, diazirines are stable at room temperature, are resistant to nucleophiles, and do not degrade under either acidic or alkaline conditions (25). With these attractive properties, diazirines have been used for the study of many signaling pathways. For example, they have been incorporated into hormones (26), epileptic drugs (27), antibiotics (28), hyperthermic drugs (29), anticancer agents (30), anesthetics (31), nucleic acids (32), amino acids (33), and lipids (34). They also have been introduced into specific molecular reporters to probe enzyme function and their binding sites such as in kinases (35), aspartic proteases (36), or metalloproteinases (37), to name a few. The nitrogen-nitrogen moiety is also intrinsically interesting, because the two atoms are usually very close in chemical shift and strongly coupled, thus suited to support a long-lived singlet state as described below.

 

Fig. 1The hyperpolarization mechanism.

(A) The precatalyst, 15N2-diazirine substrate, and p-H2 are mixed, resulting in the activated species depicted in (B). (B) Both p-H2 and the free 15N2-diazirine [2-cyano-3-(D3 methyl-15N2-diazirine)-propanoic acid] are in reversible exchange with the catalytically active iridium complex. The catalyst axial position is occupied by IMes [1,3-bis(2,4,6-trimethylphenyl)-imidazolium] and Py (pyridine) as nonexchanging ligands. The structure shown is a local energy minimum of the potential energy surface based on all-electron DFT calculations and the dispersion-corrected PBE density functional. In the complex, hyperpolarization is transferred from the parahydrogen (p-H2)–derived hydrides to the 15N nuclei (white, hydrogen; gray, carbon; blue, nitrogen; red, oxygen).

Density functional theory calculations shed light on polarization transfer catalyst

The Ir complex conformation shown in Fig. 1B was determined by all-electron density functional theory (DFT) calculations [semilocal Perdew-Burke-Ernzerhof (PBE) functional (38), corrected for long-range many-body dispersion interactions (39), in the FHI-aims software package (40, 41); see the Supplementary Materials for details]. The calculations indicate a η1 single-sided N attachment rather than η2-N=N attachment of the diazirines. In the Ir complex, hyperpolarization is transferred from p-H2 gas (~92% para-state, 7.5 atm) to the 15N2-diazirine. Both p-H2 and substrate are in reversible exchange with the central complex, which results in continuous pumping of hyperpolarization: p-H2 is continually refreshed and hyperpolarization accumulated on the diazirine substrate.

An alternate polarization transfer catalyst is introduced

As opposed to the traditional [Ir(COD)(IMes)(Cl)] catalyst (18), the synthesized [Ir(COD)(IMes)(Py)][PF6] results in a pyridine ligand trans to IMes, improving our hyperpolarization levels by a factor of ~3 (see the Supplementary Materials). We have found that this new approach, which avoids competition from added pyridine, makes it possible to directly hyperpolarize a wide variety of different types of15N-containing molecules (and even 13C). However, diazirines represent a particularly general and interesting class of ligands for molecular tags and are the focus here.

As depicted in Fig. 2A, the hyperpolarization proceeds outside the high-field NMR magnet at low magnetic fields, enabling SABRE-SHEATH directly targeting 15N nuclei (22). To establish the hyperpolarization, we bubble p-H2 for ~5 min at the adequate field. Then, the sample is transferred into the NMR magnet within ~10 s, and 15N2 signal detection is performed with a simple 90° pulse followed by data acquisition.

 

Fig. 2Experimental and spectral distinction between magnetization and singlet spin order.

(A) Experimental procedure. The sample is hyperpolarized by bubbling parahydrogen (p-H2) through the solution in the NMR tube for 5 min and subsequently transferred into the high-field magnet for detection. If the hyperpolarization/bubbling is performed in a magnetic shield at ~6 mG, z-magnetization is created (black). If the hyperpolarization/bubbling is performed in the laboratory field anywhere between ~0.1 and ~1 kG, singlet order is created (blue). (B) Z-magnetization and singlet spin order can easily be distinguished based on their spectral appearance. a.u., arbitrary units. Z-magnetization produces an in-phase quartet (black). Singlet order gives an anti-phase quartet (blue). The spin system parameters for the 15N2 two-spin system are JNN = 17.3 Hz and Δδ = 0.58 parts per million.

Two types of hyperpolarized states can be created: Magnetization and singlet order

We create two different types of hyperpolarization on the 15N2-diazirine. We can hyperpolarize traditional z-magnetization, which corresponds to nuclear spins aligned with the applied magnetic field and is associated with pure in-phase signal as illustrated with the black trace in Fig. 2B. Alternatively, we can hyperpolarize singlet order on the 15N2-diazirine, which corresponds to an anti-aligned spin state, with both spins pointing in opposite directions, entangled in a quantum mechanically “hidden” state. This hidden singlet order is converted into a detectable state when transferred to a high magnetic field and associated with the anti-phase signal illustrated by the blue trace in Fig. 2B (see the Supplementary Materials for details). The difference in symmetry of z-magnetization and singlet order leads to differences in signal decay rates; z-magnetization is directly exposed to all NMR relaxation mechanisms and is often associated with shorter signal lifetimes, which may impede molecular tracking on biologically relevant time scales. Singlet order, on the other hand, is protected from many relaxation mechanisms because it has no angular momentum (4252) and can therefore exhibit much longer lifetimes, enabling hour-long molecular tracking.

The type of hyperpolarized state is selected by the magnetic field

We can control which type of hyperpolarization we create by choosing the appropriate magnetic fields for the bubbling process. Z-magnetization is created in the SABRE-SHEATH mode at low magnetic fields inside a magnetic shield (2224, 53, 54). This behavior is explained by resonance conditions for hyperpolarizing magnetization versus singlet order that we derive in the Supplementary Materials. The condition for creating magnetization, νH − νN = |JHH ± JNN|, is field-dependent in the NMR frequencies, νH and νN, and field-independent in the J-couplings. Accordingly, hyperpolarized magnetization is created at a magnetic field where the frequency difference matches the J-couplings. This magnetic field is ~6 mG, which is obtained by using JHH = −10 Hz, JNN = −17.3 Hz, γ1H = 4.2576 kHz/G, and γ15N = −0.4316 kHz/G (see the Supplementary Materials). The theoretical prediction of 6 mG matches the experimental maximum for hyperpolarized z-magnetization illustrated by the blue data in Fig. 3A.

………

 

The demonstrated hyperpolarization lifetimes, combined with the ease of hyperpolarization in these broadly applicable biomolecular tags, may establish a paradigm shift for biomolecular sensing and reporting in optically opaque tissue. The demonstrated lifetimes even exceed lifetimes of some common radioactive tracers used in positron emission tomography (PET) (for example, 11C, 20.3 min). However, unlike PET, MR is exquisitely sensitive to chemical transformations and does not use ionizing radiation (such that, for example, daily progression monitoring of disease is easily possible). The presented work may allow direct access to biochemical mechanisms and kinetics in optically opaque media. We therefore envision tracking subtle biochemical processes in vitro with unprecedented NMR sensitivities as well as real-time in vivo biomolecular imaging with hyperpolarized diazirines.

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3D DNA Images  Nanoscale design of printed vascular tissue

Curator: Larry H. Bernstein, MD, FCAP

 

New Detailed 3D DNA Images Could Aid Nanoscale Designs

This video shows techniques that scientists used to produce 3-D reconstructions of shape fluctuations in double-helix DNA segments attached to gold nanoparticles [Lei Zhang, Dongsheng Lei, Jessica M. Smith, Meng Zhang, Huimin Tong, Xing Zhang, Zhuoyang Lu, Jiankang Liu, A. Paul Alivisatos, and Gang “Gary” Ren]

 

Flexible double-helix DNA segments connected to gold nanoparticles are revealed from the 3-D density maps (purple and yellow) reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography or IPET. Projections of the structures are shown in the background grid.[Berkeley Lab]

 

The general chemical structure of the DNA helix was described by James Watson and Francis Crick in 1953. Over the years that followed, scientists intensely studied the molecular structure of DNA to understand its behaviorin vivo and to exploit its unique properties for nanotechnology purposes.

Now, an international team of scientists working at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images from individual double-helix DNA segments attached at either end to gold nanoparticles. The images detail the flexible structure of the DNA segments, which appear as nanoscale jump ropes.

Using a cutting-edge electron microscopy (EM) technique, called individual-particle electron tomography (IPET), the researchers were able to visualize the shapes of the coiled DNA strands, which were sandwiched between polygon-shaped gold nanoparticles, and reconstruct high-resolution 3D images. The EM technique was coupled with a protein-staining process and sophisticated software that provided structural details to the scale of approximately 2 nanometers (two billionths of a meter).

“We had no idea about what the double-strand DNA would look like between the nanogold particles,” noted senior study author Gang Ren, Ph.D., staff scientist in the Molecular Foundry at Berkeley Lab. “This is the first time for directly visualizing an individual double-strand DNA segment in 3-D.”

The findings from this study were published recently in Nature Communications in an article entitled “Three-Dimensional Structural Dynamics and Fluctuations of DNA-Nanogold Conjugates by Individual-Particle Electron Tomography.”

Dr. Ren and his colleagues hope their unique imaging technique will aid in the use of DNA segments as building blocks for molecular devices that function as nanoscale drug-delivery systems, markers for biological research, and components for computer memory and electronic devices. Additionally, the research team speculates that the new method could also lead to images of important disease-relevant proteins that have proven elusive for other imaging techniques and of the assembly process that forms DNA from separate, individual strands.

The Berkeley Lab scientists flash froze samples to preserve their structure for study with cryo-EM imaging. The distance between the two gold particles in individual samples varied from 20 to 30 nanometers based on different shapes observed in the DNA segments. They then collected a series of tilted images of the stained objects and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique. They gathered a dozen confirmations for the samples and found the DNA shape variations were consistent with those measured in the flash-frozen cryo-EM samples.

While the 3D reconstructions show the basic nanoscale structure of the samples, the investigators are looking at the next steps, which will be to work on improving the resolution to the subnanometer scale.

“Even in this current state we begin to see 3D structures at 1- to 2-nanometer resolution,” Dr. Ren explained. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, Dr. Ren noted that researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami”—with the hope of building and better characterizing nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas of the body.

“DNA is easy to program, synthesize, and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” Dr. Ren stated. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”

 

Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

Lei ZhangDongsheng LeiJessica M. Smith, …., Jiankang LiuA. Paul Alivisatos & Gang Ren
Nature Communications7,Article number:11083
           doi:10.1038/ncomms11083

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Organic–inorganic-hybridized nanocrystals are a valuable class of new materials that are suitable for addressing many emerging challenges in biological and material sciences1, 2. Nanogold and quantum dot conjugates have been used extensively as biomolecular markers3, 4, whereas DNA base pairing has directed the self-assembly of discrete groupings and arrays of organic and inorganic nanocrystals in the formation of a network solid for electronic devices and memory components5. Discretely hybridized gold nanoparticles conjugated to DNA were developed as a molecular ruler to detect sub-nanometre distance changes via plasmon-coupling-mediated variations in dark-field light scattering3, 6. For many of these applications, it is desirable to obtain nanocrystals functionalized with discrete numbers of DNA strands7, 8. In all of these circumstances, the soft components can fluctuate, and the range of these structural deviations have not previously been determined with a degree of rigour that could help influence the future design and use of these assemblies.

Conformational flexibility and dynamics of the DNA-nanogold conjugates limit the structural determination by X-ray crystallography, nuclear magnetic resonance spectroscopy and single-particle cryo-electron microscopic (cryo-EM) reconstruction because they do not crystallize, are not sufficiently small for nuclear magnetic resonance studies and cannot be classified into a limited number of classes for single-particle EM reconstruction. In addition, three-dimensional (3D) structure averaged from tens of thousands of different macromolecular particles obtained without prior knowledge of the macromolecular structural flexibility could result in an absence of flexible domains upon using the single-particle reconstruction method, for example, two ankyrin repeated regions of TRPV1 were absent in its atomic resolution 3D density map9.

A fundamental experimental solution to reveal the structure of a flexible macromolecule should be based on the determination of each individual macromolecule’s structure10. Electron tomography (ET) provides high-resolution images of a single object from a series of tilted viewing angles11. ET has been applied to reveal the 3D structure of a cell section and an individual bacterium at nanometre-scale resolution12. However, reconstruction from an individual macromolecule at an intermediate resolution (1–3nm) remains challenging due to small molecular weight and low image contrast. Although, the first 3D map of an individual macromolecule, a fatty acid synthetase molecule, was reconstructed from negative-staining (NS) ET by Hoppe et al.13, serious doubts have been raised regarding the validity of this structure14, as this molecule received a radiation dose hundreds of times greater than the reported damage threshold15. Recently, we investigated the possibility based on simulated and real experimental NS and cryo-ET images10. We showed that a single-protein 3D structure at an intermediate resolution (1–3nm) is potentially achieved using our proposed individual-particle ET (IPET) method10, 16, 17, 18. IPET, an iterative refinement process using automatically generated dynamic filters and soft masks, requires no pre-given initial model, class averaging or lattice, but can tolerate small tilt errors and large-scale image distortion via decreasing the reconstruction image size to reduce the negative effects on 3D reconstruction. IPET allows us to obtain a ‘snapshot’ single-molecule 3D structure of flexible proteins at an intermediate resolution, and can be even used to reveal the macromolecular dynamics and fluctuation17.

Here we use IPET, cryo-EM and our previously reported optimized NS (OpNS)19, 20 techniques to investigate the morphology and 3D structure of hybridized DNA-nanogold conjugates. These conjugates were self-assembled from a mixture of two monoconjugates, each consisting of 84-bp single-stranded DNA and a 5-nm nanogold particle. The dimers were separated by anion-exchange high-performance liquid chromatography (HPLC) and agarose gel electrophoresis as potential substrates in plasmon-coupling experiments. By OpNS-ET imaging and IPET 3D reconstruction, we reconstruct a total of 14 density maps at a resolution of ~2nm from 14 individual double-stranded DNA (dsDNA)-nanogold conjugates. Using these maps as constraints, we derive 14 conformations of dsDNA by projecting a standard flexible dsDNA model onto the observed maps using molecular dynamics (MD) simulations. The variation of the conformations was largely consistent with that from liquid solution, and suggests that the IPET approach provides a most complete experimental determination of flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by this experiment can be expected to occur in a broad range of DNA-assembled nanostructures and flexible proteins.

 

Although the direct imaging of dsDNA has been previously reported using heavy metal shadowing32, 33 and NS methods34, 35, 36, to the best of our knowledge, the 3D structure of an individual dsDNA strand has not previously been achieved. It has been thought that individual dsDNA would be destroyed under the high energy of the electron beam before a 3D reconstruction, or even a 2D image, is able to be achieved. Our NS tilt images showing fibre-shaped dsDNA bridging two conjugated nanogold particles demonstrated that the dsDNA can in fact be directly visualized using EM, which is consistent with the recently reported single-molecule DNA sequencing technique via TEM36. The resolutions of our density maps ranged from ~14 to ~23Å, demonstrating that an intermediate-resolution 3D structure can be obtained for each individual macromolecule. This capability is consistent with our earlier report of a ~20-Å resolution 3D reconstruction of an individual IgG1 antibody using the same approach16, 17.

Notably, a total dose of ~2,000eÅ−2 used in our ET data acquisition is significantly above the limitation conventionally used in cryo-EM (~80–100eÅ−2), which can be suspected to have certain artefact from radiation damage. In cryo-EM, the radiation damage could cause sample bubbling, deformation and knockout effects; in NS, only the knockout phenomena is often observed, in which the protein is surrounded by heavy atoms that were kicked out by electron beam. Since the sample was coated with heavy metal atoms and were dried in air, the bubbling and deformation phenomena were not usually observed. The heavy metal atoms that coat the surface of the biomolecule can provide a much higher electron scattering than from a biomolecule only inside lighter atoms. The scattering is sufficiently high to provide enough image contrast at our 120-kV high tension; thus, a further increase to the scattering ability by reducing the high tension to 80kV may not be necessary for this NS sample. In addition, the heavy atoms can provide more radiation resistance and allow the sample to be imaged under a higher dose condition. The exact dose limitation for NS is still unknown. The radiation damage related artefact in NS samples is knockout, which could reduce the image contrast and lower the tilt image alignment accuracy and 3D reconstruction resolution. In our study, a total dose of 2,000eÅ−2 did not cause any obvious knockout phenomena, but provides a sufficiently high contrast for the otherwise barely visible DNA conformations in each tilt series. The direct confirmation of visible DNA in each tilt image is essentially important to us to validate each 3D reconstruction, especially considering this relatively new approach.

Our 3D reconstruction algorithm used an ab initio real-space reference-projection match iterative algorithm to correct the centres of each tilt images, in which the equal tilt angle step for 3D reconstruction of a low contrast and asymmetric macromolecule was used. This method is different from recently reported Fourier-based iterative algorithm, termed equally sloped tomography, in which the pseudo-polar fast Fourier transform, the oversampling method and internal lattice of a targeted nanoparticle are used to achieve 3D reconstruction at atomic resolution37.

It is generally challenging to achieve visualization and 3D reconstruction on an individual, small and asymmetric macromolecule by other conventional methods; our method demonstrated its capability for 3D reconstruction of 52kDa 84-bp dsDNA through these studies: IgG1 antibody 3D structural fluctuation17, peptide-induced conformational changes on flexible IgG1 antibody5, floppy liposome surface binding with 53-kDa proteins38, all of which suggest that this method could be used to serve the community as a novel tool for studying flexible macromolecular structures, dynamics and fluctuations of proteins, and for catching the intermediate 3D structure of protein assembling.

DNA-based self-assembling materials have been developed for use in materials science and biomedical research, such as DNA origami designed for targeted drug delivery. The structure, design and control require feedback from the 3D structure, which could validate the design hypothesis, optimize the synthesis protocol and improve the reproducible capability, while even providing insight into the mechanism of DNA-mediated assembly.

 

Printing Vascular Tissue

from Wyss Institute for Biologically Inspired Engineering at Harvard University

http://www.labtube.tv/video/printing-vascular-tissue

Printing vessel vasculature is essential for sustaining functional living tissues. Until now, bioengineers have had difficulty building thick tissues, lacking a method to embed vascular networks. A 3D bioprinting method invented at the Wyss Institute and Harvard SEAS embeds a grid of vasculature into thick tissue laden with human stem cells and connective matrix. Printed within a custom-made housing, this method can be used to create tissue of any shape. Once printed, an inlet and outlet own opposite ends are perfused with fluids, nutrients, and cell growth factors, which control stem cell differentiation and sustain cell functions. By flowing growth factors through the vasculature, stem cells can be differentiated into a variety of tissue cell types. This vascularized 3D printing process could open new doors to tissue replacement and engineering. Footage credit: David KA.S. Gladman, E. Matsumoto, L.K. Sanders, and J.A. Lewis / Wyss Institute at Harvard University For more information, please visit:

Scaling up tissue engineering

http://wyss.harvard.edu/viewpressrelease/250/

In this video, the Wyss Institute and Harvard SEAS team uses a customizable 3D bioprinting method to build a thick vascularized tissue structure comprising human stem cells, collective matrix, and blood vessel endothelial cells. Their work sets the stage for advancement of tissue replacement and tissue engineering techniques. Credit: Lewis Lab, Wyss Institute at Harvard University

view-source:https://player.vimeo.com/video/157497209

Bioprinting technique creates thick 3D tissues composed of human stem cells and embedded vasculature, with potential applications in drug testing and regenerative medicine

(CAMBRIDGE, Massachusetts)  — A team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School for Engineering and Applied Sciences (SEAS) has invented a method for 3D bioprinting thick vascularized tissue constructs composed of human stem cells, extracellular matrix, and circulatory channels lined with endothelial blood vessel cells. The resulting network of vasculature contained within these deep tissues enables fluids, nutrients and cell growth factors to be controllably perfused uniformly throughout the tissue. The advance is reported March 7 in the journal Proceedings of the National Academy of Sciences.

“This latest work extends the capabilities of our multi-material bioprinting platform to thick human tissues, bringing us one step closer to creating architectures for tissue repair and regeneration,” says Wyss Core Faculty member Jennifer A. Lewis, Sc.D., senior author on the study, who is also the Hansörg Wyss Professor of Biologically Inspired Engineering at SEAS.

Three-dimensional bioprinting of thick vascularized tissues

David B. Koleskya,1Kimberly A. Homana,1Mark A. Skylar-Scotta,1, and Jennifer A. Lewisa,2     aSchool of Engineering and Applied Sciences, Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138
PNAS  2016; 113(12): 3179–3184   http://dx.doi.org:/10.1073/pnas.1521342113

Significance

Current tissue manufacturing methods fail to recapitulate the geometry, complexity, and longevity of human tissues. We report a multimaterial 3D bioprinting method that enables the creation of thick human tissues (>1 cm) replete with an engineered extracellular matrix, embedded vasculature, and multiple cell types. These 3D vascularized tissues can be actively perfused with growth factors for long durations (>6 wk) to promote differentiation of human mesenchymal stem cells toward an osteogenic lineage in situ. The ability to construct and perfuse 3D tissues that integrate parenchyma, stroma, and endothelium is a foundational step toward creating human tissues for ex vivo and in vivo applications.

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

The ability to manufacture human tissues that replicate the essential spatial (1), mechanochemical (2, 3), and temporal aspects of biological tissues (4) would enable myriad applications, including 3D cell culture (5), drug screening (6, 7), disease modeling (8), and tissue repair and regeneration (9, 10). Three-dimensional bioprinting is an emerging approach for creating complex tissue architectures (10, 11), including those with embedded vasculature (1215), that may address the unmet needs of tissue manufacturing. Recently, Miller et al. (15) reported an elegant method for creating vascularized tissues, in which a sacrificial carbohydrate glass is printed at elevated temperature (>100 °C), protectively coated, and then removed, before introducing a homogeneous cell-laden matrix. Kolesky et al. (14) developed an alternate approach, in which multiple cell-laden, fugitive (vasculature), and extracellular matrix (ECM) inks are coprinted under ambient conditions. However, in both cases, the inability to directly perfuse these vascularized tissues limited their thickness (1–2 mm) and culture times (<14 d). Here, we report a route for creating thick vascularized tissues (≥1 cm) within 3D perfusion chips that provides unprecedented control over tissue composition, architecture, and microenvironment over several weeks (>6 wk). This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

Central to the fabrication of thick vascularized tissues is the design of biological, fugitive, and elastomeric inks for multimaterial 3D bioprinting. To satisfy the concomitant requirements of processability, heterogeneous integration, biocompatibility, and long-term stability, we first developed printable cell-laden inks and castable ECM based on a gelatin and fibrinogen blend (16). Specifically, these materials form a gelatin–fibrin matrix cross-linked by a dual-enzymatic, thrombin and transglutaminase (TG), strategy (Fig. 1and SI Appendix, Fig. S1). The cell-laden inks must facilitate printing of self-supporting filamentary features under ambient conditions as well as subsequent infilling of the printed tissue architectures by casting without dissolving or distorting the patterned construct (Fig. 1A). The thermally reversible gelation of the gelatin–fibrinogen network enables its use in both printing and casting, where gel and fluid states are required, respectively (SI Appendix, Fig. S2). Thrombin is used to rapidly polymerize fibrinogen (17), whereas TG is a slow-acting Ca2+-dependent enzymatic cross-linker that imparts the mechanical and thermal stability (18) needed for long-term perfusion. Notably, the cell-laden ink does not contain either enzyme to prevent polymerization during printing. However, the castable matrix contains both thrombin and TG, which diffuse into adjacent printed filaments, forming a continuous, interpenetrating polymer network, in which the native fibrillar structure of fibrin is preserved (SI Appendix, Fig. S3). Importantly, our approach allows arbitrarily thick tissues to be fabricated, because the matrix does not require UV curing (19), which has a low penetration depth in tissue (20) and can be readily expanded to other biomaterials, including fibrin and hyaluronic acid (SI Appendix, Fig. S4).

 

Fig. 1.

Fig. 1.

Three-dimensional vascularized tissue fabrication. (A) Schematic illustration of the tissue manufacturing process. (i) Fugitive (vascular) ink, which contains pluronic and thrombin, and cell-laden inks, which contain gelatin, fibrinogen, and cells, are printed within a 3D perfusion chip. (ii) ECM material, which contains gelatin, fibrinogen, cells, thrombin, and TG, is then cast over the printed inks. After casting, thrombin induces fibrinogen cleavage and rapid polymerization into fibrin in both the cast matrix, and through diffusion, in the printed cell ink. Similarly, TG diffuses from the molten casting matrix and slowly cross-links the gelatin and fibrin. (iii) Upon cooling, the fugitive ink liquefies and is evacuated, leaving behind a pervasive vascular network, which is (iv) endothelialized and perfused via an external pump. (B) HUVECs growing on top of the matrix in 2D, (C) HNDFs growing inside the matrix in 3D, and (D) hMSCs growing on top of the matrix in 2D. (Scale bar: 50 µm.) (E and F) Images of printed hMSC-laden ink prepared using gelatin preprocessed at 95 °C before ink formation (E) as printed and (F) after 3 d in the 3D printed filament where actin (green) and nuclei (blue) are stained. (G) Gelatin preprocessing temperature affects the plateau modulus and cell viability after printing. Higher temperatures lead to lower modulus and higher HNDF viability postprinting. (H) Photographs of interpenetrated sacrificial (red) and cell inks (green) as printed on chip. (Scale bar: 2 mm.) (I) Top-down bright-field image of sacrificial and cell inks. (Scale bar: 50 µm.). (J–L) Photograph of a printed tissue construct housed within a perfusion chamber (J) and corresponding cross-sections (K and L). (Scale bars: 5 mm.)

To construct thick, vascularized tissues within 3D perfusion chips, we coprinted cell-laden, fugitive, and silicone inks (Fig. 1 H and I). First, the silicone ink is printed on a glass substrate and cured to create customized perfusion chips (Movie S1 and SI Appendix, Fig. S1). Next, the cell-laden and fugitive inks are printed on chip, and then encapsulated with the castable ECM (Fig. 1 J–L and Movie S2). The fugitive ink, which defines the embedded vascular network, is composed of a triblock copolymer [i.e., polyethylene oxide (PEO)–polypropylene oxide (PPO)–PEO]. This ink can be removed from the fabricated tissue upon cooling to roughly 4 °C, where it undergoes a gel-to-fluid transition (14, 23). This process yields a pervasive network of interconnected channels, which are then lined with HUVECs. The resulting vascularized tissues are perfused via their embedded vasculature on chip over long time periods using an external pump (Movie S3) that generates smooth flow over a wide range of flow rates (24).

http://static-movie-usa.glencoesoftware.com/webm/10.1073/609/83291359a6e4390b2232b577cb8c8742d967acef/pnas.1521342113.sm03.webm

Movie S3.

Fluorescent microscopy video of different perfusion rates through the embedded vasculature within the printed 3D tissue microenvironments.

http://www.pnas.org/content/113/12/3179/F2.medium.gif

Fig. 2.

Three-dimensional vascularized tissues remain stable during long-term perfusion. (A) Schematic depicting a single HUVEC-lined vascular channel supporting a fibroblast cell-laden matrix and housed within a 3D perfusion chip. (B and C) Confocal microscopy image of the vascular network after 42 d, CD-31 (red), vWF (blue), and VE-Cadherin (magenta). (Scale bars: 100 µm.) (D) Long-term perfusion of HUVEC-lined (red) vascular network supporting HNDF-laden (green) matrix shown by top-down (Left) and cross-sectional confocal microscopy at 45 d (Right). (Scale bar: 100 µm.) (E) Quantification of barrier properties imparted by endothelial lining of channels, demonstrated by reduced diffusional permeability of FITC-dextran. (F) GFP-HNDF distribution within the 3D matrix shown by fluorescent intensity as a function of distance from vasculature.

http://static-movie-usa.glencoesoftware.com/webm/10.1073/609/83291359a6e4390b2232b577cb8c8742d967acef/pnas.1521342113.sm04.webm

Movie S4.

Confocal microscopy video of cross-section through vascularized tissue after 45 d of perfusion.

To explore emergent phenomena in complex microenvironments, we created a heterogeneous tissue architecture (>1 cm thick and 10 cm3 in volume) by printing a hMSC-laden ink into a 3D lattice geometry along with intervening in- and out-of-plane (vertical) features composed of fugitive ink, which ultimately transform into a branched vascular network lined with HUVECs. After printing, the remaining interstitial space is infilled with an HNDF-laden ECM (Fig. 3A) to form a connective tissue that both supports and binds to the printed stem cell-laden and vascular features. In this example, fibroblasts serve as model cells that surround the heterogeneously patterned stem cells and vascular network. These model cells could be replaced with either support cells (e.g., immune cells or pericytes) or tissue-specific cells (e.g., hepatocytes, neurons, or islets) in future embodiments. The embedded vascular network is designed with a single inlet and outlet that provides an interface between the printed tissue and the perfusion chip. This network is symmetrically branched to ensure uniform perfusion throughout the tissue, including deep within its core. In addition to providing transport of nutrients, oxygen, and waste materials, the perfused vasculature is used to deliver specific differentiation factors to the tissue in a more uniform manner than bulk delivery methods, in which cells at the core of the tissue are starved of factors (25). This versatile platform (Fig. 3A) is used to precisely control growth and differentiation of the printed hMSCs. Moreover, both the printed cellular architecture and embedded vascular network are visible macroscopically with this thick tissue (Fig. 3B).

Fig. 3.

Fig. 3.

Osteogenic differentiation of thick vascularized tissue. (A) Schematic depicting the geometry of the printed heterogeneous tissue within the customized perfusion chip, wherein the branched vascular architecture pervades hMSCs that are printed into a 3D lattice architecture, and HNDFs are cast within an ECM that fills the interstitial space. (B) Photographs of a printed tissue construct within and removed from the customized perfusion chip. (C) Comparative cross-sections of avascular tissue (Left) and vascularized tissue (Right) after 30 d of osteogenic media perfusion with alizarin red stain showing location of calcium phosphate. (Scale bar: 5 mm.) (D) Confocal microscopy image through a cross-section of 1-cm-thick vascularized osteogenic tissue construct after 30 d of active perfusion and in situ differentiation. (Scale bar: 1.5 mm.) (E) Osteocalcin intensity across the thick tissue sample inside the red lines shown in C. (F) High-resolution image showing osteocalcin (purple) localized within hMSCs, and they appear to take on symmetric osteoblast-like morphologies. (Scale bar: 100 µm.) After 30 d (Gand H), thick tissue constructs are stained for collagen-I (yellow), which appears to be localized near hMSCs. (Scale bars: 200 µm.) (I) Alizarin red is used to stain calcium phosphate deposition, and fast blue is used to stain AP, indicating tissue maturation and differentiation over time. (Scale bar: 200 µm.)

In summary, thick, vascularized human tissues with programmable cellular heterogeneity that are capable of long-term (>6-wk) perfusion on chip have been fabricated by multimaterial 3D bioprinting. The ability to recapitulate physiologically relevant, 3D tissue microenvironments enables the exploration of emergent biological phenomena, as demonstrated by observations of in situ development of hMSCs within tissues containing a pervasive, perfusable, endothelialized vascular network. Our 3D tissue manufacturing platform opens new avenues for fabricating and investigating human tissues for both ex vivo and in vivo applications.

 

Steve Dufourny Hello Mr Bernstein, it is relevant.I have a model with quantum sphères and spherical volumes correlated with my theory of spherisation in theoretical physics(in a very simple resume quant sphères…..spherisation encodings…….Cosmol sphères…….Universal sphères with a central BH)more two équations about matter and energy and sphères.The quantizations and properties can be computed.The adn, amino acids ,protiens INE ASE are the keys.I search the main gravitationalcodes.It is more far than our standard model in logic.Regards

 

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